Use of phospho-akt as a biomarker of drug response

ABSTRACT

Use of phospho-Akt as a biomarker for predicting the response, such as resistance, to a compound, wherein phospho-Akt is Akt that has been phosphorylated on one or more residues, with the proviso that for Akt1, Akt2, and Akt3 the designation phospho-Akt is used to indicate phosphorylation at a site other than T308, T309 or T305 respectively, wherein the compound is a compound of general formula (I) wherein R represents phenyl, thienyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently selected from alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted amino wherein the two substituents on nitrogen form together with the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein pyridinyl is optionally substituted by lower alkoxy, amino or halogen; X represents a group C═Y, wherein Y stands for oxygen or nitrogen substituted by hydroxy or lower alkoxy; R 1  represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R 2 , R 3  and R 6  represent hydrogen; R 4  and R 5 , independently of each other, represent hydrogen, lower alkyi or lower alkoxy; or R 4  and R 5  together represent methylenedioxy; and pharmaceutically acceptable derivatives thereof; or wherein R represents phenyl or pyridinyl wherein phenyl is optionally substituted by one or two substituents independently selected from alkyi, halo-lower alkyi, hydroxy-lower alkyi, lower alkoxy-lower alkyi, acyloxy-lower alkyi, phenyl, hydroxy, lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino, dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino, substituted amino wherein the two substituents on nitrogen form together with the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, formyl, cyano, halogen, and nitro; and wherein two adjacent substituents are methylenedioxy; and wherein pyridinyl is optionally substituted by lower alkoxy, amino or halogen; X represents oxygen; R′ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R 2 , R 3  and R 6  represent hydrogen; R 4  and R 5 , independently of each other, represent hydrogen, lower alkyl or lower alkoxy; or R 4  and R 5  together represent methylenedioxy; and pharmaceutically acceptable derivatives thereof. Methods of treatment of neoplastic and autoimmune diseases with these compounds are also disclosed.

The present invention relates to use of phospho-Akt as a biomarker forpredicting the response of a disease, such as a neoplastic or autoimmunedisease, preferably cancer, to a compound of general formula I, such as3-(4-{1-[2-(4-amino-phenyl)-2-oxo-ethyl]-1H-benzoimidazol-2-yl}-furazan-3-ylamino)-propionitrile (BAL27862). Inother aspects it relates to methods and kits, as well as methods oftreatment involving the use of the biomarker.

Microtubules are one of the components of the cell cytoskeleton and arecomposed of heterodimers of alpha- and beta-tubulin. Agents that targetmicrotubules are among the most effective cytotoxic chemotherapeuticagents having a broad spectrum of activity. Microtubule destabilisingagents (e.g. the vinca-alkaloids such as vincristine, vinblastine andvinorelbine) are used for example in the treatment of several types ofhematologic malignancies, such as lymphoblastic leukaemia and lymphoma,as well as solid tumours, such as lung cancer. Microtubule stabilisingagents (e.g. the taxanes such as paclitaxel, docetaxel) are used forexample in the treatment of solid tumours, including breast, lung andprostate cancer.

However resistance to these known microtubule targeting agents canoccur. The resistance can either be inherent or can be acquired afterexposure to these agents. Such resistance therefore impacts patientsurvival rates, as well as choices of treatment regimes. Severalpotential mechanisms of resistance have been identified, and includedefects in the microtubule targets, such as elevated levels ofbeta-tubulin subtype III and acquired mutations in beta-tubulin subtypeI that are known to reduce taxane binding. Furthermore, defects in othercell proteins have been suggested to be associated with resistance tocertain microtubule targeting agents, such as overexpression of theefflux pump P-glycoprotein (P-gp, also known as multi-drug resistanceprotein 1 or MDR1). Such factors may then be used as biomarkers ofresistance to these conventional microtubule targeting agents.

A relatively recently discovered class of microtubule destabilisingagents are compounds encompassed by the formula given below:

whereinR represents phenyl, thienyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacentsubstituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents a group C═Y, wherein Y stands for oxygen or nitrogensubstituted by hydroxy or lower alkoxy;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable salts thereof;or whereinR represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, formyl, cyano, halogen, and nitro; and wherein twoadjacent substituents are methylenedioxy; and wherein pyridinyl isoptionally substituted by lower alkoxy, amino or halogen;X represents oxygen;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable salts thereof;and wherein the prefix lower denotes a radical having up to andincluding a maximum of 7, especially up to and including a maximum of 4carbon atoms.

These compounds are disclosed in WO2004/103994 A1, which is incorporatedby cross-reference herein. These compounds have been shown to arresttumour cell proliferation and induce apoptosis.

The synthesis of compounds of formula I is described in WO2004/103994A1, in general on pages 29-35, and specifically on pages 39-55, whichare incorporated herein by cross-reference. They may be prepared asdisclosed or by an analogous method to the processes described therein.

One compound falling within this class, known as BAL27862, and shown inWO2004/103994 A1 as example 58, and specifically incorporated byreference herein, has the structure and chemical name given below:

Chemical name:3-(4-{1-[2-(4-Amino-phenyl)-2-oxo-ethyl]-1H-benzoimidazol-2-yl}-furazan-3-ylamino)-propionitrile;or herein as Compound A.

Further compounds exemplified in WO2004/103994 A1 as examples 50 and 79respectively, and also specifically incorporated by cross-referenceherein, have the structures and chemical names given below:

Chemical name:2-[2-(4-Amino-furazan-3-yl)-benzoimidazol-1-yl]-1-(4-amino-phenyl)-ethanone;or herein as Compound B and

Chemical name:3-(4-{1-[2-(6-Amino-pyridin-3-yl)-2-oxo-ethyl]-1H-benzoimidazol-2-yl}-furazan-3-ylamino)-propionitrile;or herein as Compound C.

BAL27862 has activity across a broad panel of experimental, solid tumourxenograft models. Moreover, activity is retained even against tumourmodels which are selected for resistance to conventional microtubuletargeting agents (including the vinca-alkaloid microtubule destabilisersand the microtubule stabilisers paclitaxel and epothilone B). BAL27862activity is not affected by over-expression of the P-gp pump in anymodels tested in vitro, nor in human mammary tumour xenografts.Additionally, BAL27862 retained its activity despite elevated levels ofbeta-tubulin subtype III and mutations in tubulin subtype I.

Hence, BAL27862 activity is not affected by a number of factors thatconfer resistance to conventional microtubule targeting agents.

Moreover, it is known that compounds of general formula I have adifferent effect on the phenotype of cells compared to other microtubuletargeting agents, including other microtubule destabilisers. Treatmentwith a compound of general formula I induces a consistent microtubulephenotype in tumour cell lines derived from a variety of organs, forexample lung, cervix and breast, as seen in FIG. 1. Staining themicrotubules in these cells with an anti-alpha-tubulin antibody showsthat rather than the mitotic spindle fibres of untreated cells, onlydot-like structures are visible in the treated cells. This same effectis also shown using Compounds C and B in FIGS. 2A and 2B respectively onthe lung cancer cell line A549. It is however very distinct from thatobserved with the conventional microtubule targeting agents vinblastine,colchicine, paclitaxel and nocodazole as seen in FIGS. 3B, 3C, 3D and 4,respectively. The microtubules were stained with an anti-alpha-tubulinantibody and the cells viewed at a 1000× magnification (FIGS. 3, 4). Forthe cells treated with BAL27862, multiple dot-like structures arevisible, whereas, in stark contrast, the other conventional drugsproduce filamentous microtubule structures, or dense microtubuleaggregate structures. These differences at the phenotypic level, atcompound doses considered optimal in terms of antiproliferative effect,indicate a difference in the mode of action at the molecular level.

Furthermore, it is known that BAL27862 elicits a dominant microtubulephenotype in the presence of the other microtubule targeting agents.Treatment with vinblastine, colchicine, paclitaxel or nocodazole aloneinduced the microtubule phenotypes characteristic of these agents (FIG.5A, 5D, 5G, 6C-6F respectively). However, combination treatment withBAL27862 for the last 4 hours resulted in disruption of thesephenotypes; despite the continued presence of vinblastine, colchicine,paclitaxel or nocodazole (FIG. 5B, 5E, 5H, 6G-6J respectively). Incontrast, treating first with BAL27862 and subsequently for 4 hours incombination with vinblastine, colchicine, paclitaxel or nocodazole hadno impact on generation of the phenotype consistent with BAL27862treatment (FIG. 5C, 5F, 5I, 6K-6N respectively).

These data all demonstrate that BAL27862 affects microtubule biology ina different manner than conventional microtubule targeting agents.

Thus, from information about conventional microtubule targeting agents,predictions cannot be made concerning if, or how, particular genes areinvolved in the action of compounds of general formula I.

An object of the present invention is to identify factors which areassociated with response to compounds of formula I or pharmaceuticallyacceptable derivatives thereof, for example to identify factorsassociated with resistance to compounds of general formula I, inparticular BAL27862 or pharmaceutically acceptable derivatives thereof,as defined below.

It has surprisingly been found that phospho-Akt may be used as abiomarker of response to treatment with a compound of general formula Ior pharmaceutically acceptable derivatives thereof, as defined below.

In one preferred embodiment of the invention, relatively highphospho-Akt levels in a tumour sample are associated with inherentresistance to BAL27862.

To date, the Akt family consists of three known genes, also referred toas isoforms, Akt1, Akt2 and Akt3. These genes are homologous at thenucleic acid level, as well as at the polypeptide sequence level. TheAkt genes are also known by a variety of alternative names, reflectingtheir discovery by different groups. The name Akt arose from itsdiscovery as the human homologue of the proto-oncogene of thetransforming retrovirus AKT8. Akt is also known as protein kinase B, orRAC (Related to A and C kinases), since these Akt proteins are closelyrelated to protein kinase A (PKA) and protein kinase C (PKC). Thus, theAkt family is known by the following synonyms c-AKT; proto-oncogenec-Akt; protein kinase B; PKB; RAC; RAC serine/threonine-protein kinase;rac protein kinase and RAC-PK. Akt1, the gene first discovered, is alsoknown as c-AKT1; PKB; PKB-alpha; PRKBA; RAC; RAC-alphaserine/threonine-protein kinase; RAC-ALPHA; RAC-PK-alpha and MGC99656.Akt2 is also known as protein kinase Akt-2; protein kinase B beta;PKBBETA; PRKBB; RAC-BETA; RAC-beta serine/threonine-protein kinase andRAC-PK-beta. Akt3 is also known as PKB-gamma; PKBG; PRKBG; RAC-gammaserine/threonine-protein kinase; RAC-gamma; RAC-PK-gamma; DKFZP434N0250and STK-2. Alternative splice transcript variants have also been foundfor some of these genes. Alternative splice transcript variants encodingdistinct isoforms have been described for Akt3, namely RAC-gammaserine/threonine-protein kinase isoform 1 and RAC-gammaserine/threonine-protein kinase isoform 2, which differ in length. Thedesignation Akt shall be used herein to encompass the three relatedproteins Akt1, Akt2, Akt3 and all the synonyms listed above, includingisoforms.

Akt may be post-translationally modified, including by phosphorylationat one or more sites. For example Akt1 is known to be able to bephosphorylated on Ser-124, Thr-308, Thr-450, and Ser-473. More than onesite may be phosphorylated simultaneously. Regulation of the function ofAkt has been particularly identified in connection with thephosphorylation of two sites: a threonine: T308 (Akt 1), T309 (Akt 2),T305 (Akt 3), and a serine: S473 (Akt 1), S474 (Akt 2), S472 (Akt 3).

Phosphoinositide dependent kinase 1 (PDK1) is thought to phosphorylatethreonine 308, while mTOR Complex 2 (mTORC2) has recently beenidentified as PDK2, as it is thought to phosphorylate serine 473 ofAkt1.

As used herein, phospho-Akt shall refer to Akt that has beenphosphorylated on one or more residues, with the proviso that for Akt1,Akt2, and Akt3 the designation phospho-Akt is used to indicatephosphorylation at a site other than T308, T309 or T305 respectively. Toclarify this further, the phospho-Akt may or may not be phosphorylatedat T308, T309 or T305 for Akt1, Akt2 and Akt3 respectively, but thedesignation phospho-Akt indicates phosphorylation at sites other thanthese. Phospho-Akt may optionally also be post-translationally modifiedin a way other than by phosphorylation.

More preferably, phospho-Akt shall refer to Akt that has beenphosphorylated on the following serine residue:

for Akt1: S473; for Akt2: S474; and for Akt3: S472.

This preferred embodiment therefore does not encompass the Akt3 encodedby RAC-gamma serine/threonine-protein kinase isoform 2, since it doesnot have a serine 472.

Protein sequences coding for human Akt1, Akt2 and Akt3 are available viaNational Center for Biotechnology Information (NCBI) accession numbers

Akt1: NP_(—)005154.2, see SEQ. ID. No. 1 (see also NP_(—)001014431 andNP_(—)001014432),

Akt2: NP_(—)001617.1, see SEQ ID No. 2 and

Akt3: NP_(—)005456.1: RAC-gamma serine/threonine-protein kinase isoform1, see SEQ ID No. 3.

One aspect of the present invention relates to use of phospho-Akt as abiomarker for predicting the response to a compound,

wherein phospho-Akt is Akt that has been phosphorylated on one or moreresidues, with the proviso that for Akt1, Akt2, and Akt3 the designationphospho-Akt is used to indicate phosphorylation at a site other thanT308, T309 or T305 respectively,

wherein the compound is a compound of general formula I,

whereinR represents phenyl, thienyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacentsubstituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents a group C═Y, wherein Y stands for oxygen or nitrogensubstituted by hydroxy or lower alkoxy;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable derivatives thereof,or whereinR represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, formyl, cyano, halogen, and nitro; and wherein twoadjacent substituents are methylenedioxy; and wherein pyridinyl isoptionally substituted by lower alkoxy, amino or halogen;X represents oxygen;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable derivatives thereof;and wherein the prefix lower denotes a radical having up to andincluding a maximum of 7, especially up to and including a maximum of 4carbon atoms.

Preferably the response may be of a disease in a subject. Alsopreferably the response may be to treatment, i.e. to treatment with thecompound of general formula I or pharmaceutically acceptable derivativesthereof.

The biomarker phospho-Akt is measured ex vivo in a sample or samplestaken from the human or animal body, preferably taken from the humanbody. The sample or samples are pre-obtained from the human or animalbody, preferably pre-obtained from the human body.

In a preferred embodiment, the invention relates to use of phospho-Aktas a biomarker for predicting the resistance of a disease in a subjectto a compound of general formula I or pharmaceutically acceptablederivatives thereof as defined above.

Preferably the pharmaceutically acceptable derivative is selected fromthe group consisting of a salt, solvate, pro-drug, salt of a pro-drug,polymorph and isomer of a compound of general formula I. Pro-drugs arepreferably ester and amides of naturally occurring amino acids, smallpeptides or pegylated hydroxy acids. More preferably, the pro-drug is anamide formed from an amino group present within the R group of thecompound of general formula I and the carboxy group of glycine, alanineor lysine.

Particularly preferably the compound is

or a pharmaceutically acceptable salt thereof, preferably ahydrochloride salt thereof, most preferably a dihydrochloride saltthereof.

Another aspect of the present invention relates to a method forpredicting the response of a disease in a subject to a compound ofgeneral formula I or pharmaceutically acceptable derivatives thereof asdefined above, comprising the steps of:

-   -   a) measuring a level of phospho-Akt as defined above in a sample        pre-obtained from the subject to obtain a value or values        representing this level; and    -   b) comparing the value or values from step a) to a standard        value or set of standard values.

Further preferably the response which is predicted is resistance.

The measuring of a level or levels of phospho-Akt is performed ex-vivoin a sample pre-obtained from the subject. Pre-obtained refers to thefact that the sample is obtained before it is subjected to any methodinvolving measuring the level of the biomarker, and pre-obtained is notto be understood as in relation to treatment.

In a preferred embodiment, a higher level of phospho-Akt in the samplefrom the subject relative to the standard value or set of standardvalues predicts resistance.

Also preferably, the disease is a neoplastic or autoimmune disease. Morepreferably the disease is cancer. Especially preferably the cancer isselected from the group consisting of breast cancer, prostate cancer,cervical cancer, gastric cancer, ovarian cancer, colorectal cancer (i.eincluding colon cancer and rectal cancer), pancreatic cancer, livercancer, brain cancer, neuroendocrine cancer, lung cancer, kidney cancer,hematological malignancies, melanoma and sarcomas. More especiallypreferably the cancer is selected from the group consisting of breastcancer, cervical cancer, gastric cancer, lung cancer, colorectal cancerand melanoma. Particularly preferably the cancer is selected from thegroup consisting of gastric cancer, lung cancer, colorectal cancer andmelanoma.

In a further aspect, the invention relates to a method of treating aneoplastic or autoimmune disease, preferably cancer, in a subject inneed thereof, comprising measuring a level of phospho-Akt as definedabove in a sample from the subject to obtain a value or valuesrepresenting this level, and treating the subject with a compound ofgeneral formula I or a pharmaceutically acceptable derivative thereof asdefined above, if the level of phospho-Akt in said sample is not higherthan a standard value or set of standard values.

In yet a further aspect, the invention relates to phospho-Akt as definedabove for use in the treatment of a neoplastic or autoimmune disease,preferably cancer, comprising measuring a level of phospho-Akt in asample from a subject to obtain a value or values representing thislevel, and treating the subject with a compound of general formula I ora pharmaceutically acceptable derivative thereof as defined above, ifthe level of phospho-Akt is not higher than a standard value or set ofstandard values.

The measuring of a level of phospho-Akt is performed ex-vivo in a samplepre-obtained from the subject.

The invention also relates in another aspect to a method of treating aneoplastic or autoimmune disease, preferably cancer, by first decreasinga level of phospho-Akt as defined above in a subject that has a samplewith a higher level of phospho-Akt compared to a standard level or setof standard levels, then treating the subject with a compound of generalformula I or a pharmaceutically acceptable derivative thereof as definedabove.

In yet another aspect the invention relates to a kit for predicting theresponse to a compound of general formula I or a pharmaceuticallyacceptable derivative thereof, as defined above, comprising reagentsnecessary for measuring the level of phospho-Akt as defined above in asample. More preferably the kit also comprises a comparator module whichcomprises a standard value or set of standard values to which the levelof phospho-Akt in the sample is compared.

More preferably the kit comprises a compound of general formula I or apharmaceutically acceptable derivative thereof as defined above. In anespecially preferred embodiment the kit comprises a compound of thefollowing formula or a pharmaceutically acceptable salt thereof

Chemical name: S-2,6-Diamino-hexanoic acid[4-(2-{2-[4-(2-cyano-ethylamino)-furazan-3-yl]-benzoimidazol-1-yl}-acetyl)-phenyl]-amide

In a particularly preferred embodiment the pharmaceutically acceptablesalt is a dihydrochloride salt.

Another further aspect of the invention relates to a device forpredicting the response to a compound of general formula I or apharmaceutically acceptable derivative thereof as defined above,comprising reagents necessary for measuring a level of phospho-Akt asdefined above in a sample and a comparator module which comprises astandard value or set of standard values to which the level ofphospho-Akt in the sample is compared.

In a preferred embodiment, the reagents in the kit or device comprise acapture reagent comprising a detector for phospho-Akt, and a detectorreagent. Especially preferably the capture reagent is an antibody. Alsopreferably, the disease is predicted to be resistant to treatment withsaid compound when phospho-Akt is higher relative to a standard value orset of standard values. In a preferred embodiment, the comparator moduleis included in instructions for use of the kit. In another preferredembodiment the comparator module is in the form of a display device.

Embodiments of the present invention will now be described by way ofexample with reference to the accompanying figures. The inventionhowever is not to be understood as limited to these embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the treatment of human tumour cell lines from differenthistotypes with 50 nM BAL27862. The microtubules of mitotic or G2/Marrested cells were stained after 24 hours treatment with 50 nM BAL27862or vehicle control.

FIGS. 1A and 1B: A549 NSCLC cells;

FIGS. 1C and 1D: HeLa cervical cancer cells;

FIGS. 1E and 1F: SKBR3 breast cancer cells

Vehicle control treatment: FIGS. 1A, 1C & 1E,

BAL27862 treatment: FIGS. 1B, 1D & 1F.

FIG. 2: Shows the treatment of A549 NSCLC cells with the Compounds B andC. The microtubules of mitotic or G2/M arrested A549 NSCLC cells werestained after 24 hours treatment with 80 nM or 20 nM of Compounds B andC, respectively. The white scale bar represents 10 micrometres.

FIG. 2A: treatment with 20 nM compound C

FIG. 2B: treatment with 80 nM compound B

FIG. 3: Shows a comparison of treatment of cells with BAL27862 comparedto conventional microtubule targeting agents. Microtubules of mitotic orG2/M arrested A549 NSCLC cells were stained after 24 hours of treatmentwith 50 nM of A: BAL27862; B: vinblastine; C: colchicine; D: paclitaxel.Stacks of images taken every 1 μm were processed by using ImageJsoftware.

FIG. 4: Shows a comparison of treatment of A549 NSCLC cells withBAL27862 compared to nocodazole. Microtubules of mitotic or G2/Marrested cells were stained after 24 h of treatment with variousconcentrations of nocodazole (B, C & D) and BAL27862 (E, F & G). A:control, B: Nocodazole 50 nM, C: Nocodazole 100 nM, D: Nocodazole 200nM, E: BAL27862 20 nM; F: BAL27862 30 nM and G: BAL27862 50 nM. Thewhite scale bar represents 10 micrometres. Representative images of themicrotubule phenotypes observed are shown.

FIG. 5: Shows a combination of treatment with BAL27862 and conventionalmicrotubule-targeting agents. Microtubules of mitotic or G2/M arrestedA549 NSCLC cells were stained after treatment for the times indicatedbelow. 50 nM BAL27862, 50 nM vinblastine, 50 nM colchicine and 25 nMpaclitaxel were used. The white scale bar represents 10 micrometres.

FIG. 5A: 24 hours vinblastine treatment;

FIG. 5B: 24 hours vinblastine treatment with the final 4 hours includingBAL27862;

FIG. 5C: 24 hours BAL27862 treatment with the final 4 hours includingvinblastine.

FIG. 5D: 24 hours colchicine treatment;

FIG. 5E: 24 hours colchicine treatment with the final 4 hours includingBAL27862;

FIG. 5F: 24 hours BAL27862 treatment with the final 4 hours includingcolchicine.

FIG. 5G: 24 hours paclitaxel treatment;

FIG. 5H: 24 hours paclitaxel treatment with the final 4 hours includingBAL27862;

FIG. 5I: 24 hours BAL27862 treatment with the final 4 hours includingpaclitaxel.

FIG. 6: Shows a combination of treatment with BAL27862 and nocodazole.Microtubules of mitotic or G2/M arrested A549 NSCLC cells were stainedafter treatment for the times indicated below. 25 nM BAL27862 andnocodazole at the concentrations indicated below were used. The whitescale bar represents 10 micrometers.

FIG. 6A: 24 hours control treatment;

FIG. 6B: 24 hours of 25 nM BAL27862 treatment;

FIG. 6C: 24 hours of 50 nM nocodazole treatment

FIG. 6D: 24 hours of 100 nM nocodazole treatment

FIG. 6E: 24 hours of 150 nM nocodazole treatment

FIG. 6F: 24 hours of 200 nM nocodazole treatment

FIG. 6G: 24 hours of 50 nM nocodazole treatment with the final 4 hoursincluding 25 nM BAL27862;

FIG. 6H: 24 hours of 100 nM nocodazole treatment with the final 4 hoursincluding 25 nM BAL27862;

FIG. 6I: 24 hours of 150 nM nocodazole treatment with the final 4 hoursincluding 25 nM BAL27862;

FIG. 6J: 24 hours of 200 nM nocodazole treatment with the final 4 hoursincluding 25 nM BAL27862;

FIG. 6K: 24 hours of 25 nM BAL27862 treatment with the final 4 hoursincluding 50 nM nocodazole;

FIG. 6L: 24 hours of 25 nM BAL27862 treatment with the final 4 hoursincluding 100 nM nocodazole;

FIG. 6M: 24 hours of 25 nM BAL27862 treatment with the final 4 hoursincluding 150 nM nocodazole;

FIG. 6N: 24 hours of 25 nM BAL27862 treatment with the final 4 hoursincluding 200 nM nocodazole.

FIG. 7: Shows protein extracts prepared from patient-derived gastriccancer (FIG. 7A), lung cancer (FIG. 7B), colorectal cancer (FIG. 7C) andmelanoma (FIG. 7D) tumours obtained from subcutaneously xenografted nudemice, and analysed by immunoblotting for phospho-Akt and Akt expression,with actin included as a loading control. Three independent tumours wereanalysed in each case (1-3). BAL27862, paclitaxel and vinblastineresistance and sensitivity of the tumour cells using an ex vivo colonyoutgrowth assay is as defined in Table 1.

FIG. 8: Shows that tumour cell phospho-Akt levels are increased in apatient-derived xenografted gastric tumour model defined as BAL27862resistant by ex vivo colony outgrowth analysis. Patient-derived tumourxenografts (maintained in nude mice) were prepared, fixed and stainedfor phospho-Akt protein expression using immunohistochemistry. BAL27862,paclitaxel and vinblastine resistance and sensitivity of the tumourcells using an ex vivo colony outgrowth assay is as defined in Table 1.

FIG. 9: shows the protein sequence of Akt1 (RAC-alphaserine/threonine-protein kinase) [Homo sapiens] (SEQ. ID. No. 1)

FIG. 10: shows the protein sequence of Akt2 (RAC-betaserine/threonine-protein kinase) [Homo sapiens] (SEQ. ID. No. 2)

FIG. 11: shows the protein acid sequence of Akt3 (RAC-gammaserine/threonine-protein kinase isoform 1) [Homo sapiens] (SEQ. ID. No.3)

DETAILED DESCRIPTION

Compounds of Formula I

The compounds according to the invention are represented by generalformula I:

whereinR represents phenyl, thienyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacentsubstituents are methylenedioxy;and wherein pyridinyl is optionally substituted by lower alkoxy, aminoor halogen;X represents a group C═Y, wherein Y stands for oxygen or nitrogensubstituted by hydroxy or lower alkoxy;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable derivatives thereof,or whereinR represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, formyl, cyano, halogen, and nitro; and wherein twoadjacent substituents are methylenedioxy; and wherein pyridinyl isoptionally substituted by lower alkoxy, amino or halogen;X represents oxygen;R¹ represents hydrogen, lower alkylcarbonyl, hydroxy-lower alkyl orcyano-lower alkyl;R², R³ and R⁶ represent hydrogen;R⁴ and R⁵, independently of each other, represent hydrogen, lower alkylor lower alkoxy;or R⁴ and R⁵ together represent methylenedioxy;and pharmaceutically acceptable derivatives thereof;and wherein the prefix lower denotes a radical having up to andincluding a maximum of 7, especially up to and including a maximum of 4carbon atoms.

Heterocyclyl designates preferably a saturated, partially saturated orunsaturated, mono- or bicyclic ring containing 4-10 atoms comprisingone, two or three heteroatoms selected from nitrogen, oxygen and sulfur,which may, unless otherwise specified, be carbon or nitrogen linked,wherein a ring nitrogen atom may optionally be substituted by a groupselected from lower alkyl, amino-lower alkyl, aryl, aryl-lower alkyl andacyl, and a ring carbon atom may be substituted by lower alkyl,amino-lower alkyl, aryl, aryl-lower alkyl, heteroaryl, lower alkoxy,hydroxy or oxo. Examples of heterocyclyl are pyrrolidinyl, oxazolidinyl,thiazolidinyl, piperidinyl, morpholinyl, piperazinyl, dioxolanyl andtetrahydropyranyl.

Acyl designates, for example, alkylcarbonyl, cyclohexylcarbonyl,arylcarbonyl, aryl-lower alkylcarbonyl, or heteroarylcarbonyl. Loweracyl is preferably lower alkylcarbonyl, in particular propionyl oracetyl.

Preferably, the compound of general formula I according to the inventionis defined as wherein R¹ is selected from the group consisting ofhydrogen, acetyl, CH₂CH₂CN and CH₂CH₂CH₂OH.

In one preferred embodiment, the compound of general formula I accordingto the invention is selected from the group consisting of:

-   4-(1-Phenacyl-1H-benzimidazol-2-yl)-furazan-3-ylamine,-   4-[1-(4-Bromophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine    oxime,-   N-{4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl}-acetamide,-   4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl)-amine,-   4-[1-(4-Chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(3-hydroxypropyl)-amine,-   4-[1-(3-Amino-4-chlorophenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine,-   4-[1-(3-Methoxy-4-methoxymethoxy-phenacyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine,    and pharmaceutically acceptable derivatives thereof.

In another preferred embodiment, the compound of general formula Iaccording to the invention is:

whereinR, Y and R¹ are defined as follows:

R Y R¹

O H

NOH H

NOMe H

O H

NOH H

NOH H

NOMe H

O H

NOH H

NOMe H

O H

NOH H

NOMe H

O H

NOMe H

O H

O H

NOH H

NOMe H

O H

NOH H

NOMe H

NOMe H

O H

O Ac

O H

O H

O H

O CH₂CH₂CN

O CH₂CH₂CN

O H

O H

O CH₂CH₂CH₂OH

O H

O CH₂CH₂CN

O H

O CH₂CH₂CN

O CH₂CH₂CN

O CH₂CH₂CN

O H

O H

O H

O H

O H

O H

O H

O H

O CH₂CH₂CN

O H

O H

O H

O H

O H

O H

O H

O H

O H

O CH₂CH₂CN

O H

O H

O CH₂CH₂CNor pharmaceutically acceptable derivatives thereof.

In yet another preferred embodiment, the compound of general formula Iaccording to the invention is selected from the group consisting of:

-   4-(1-Phenoxymethyl-1H-benzimidazol-2-yl)-furazan-3-ylamine,-   4-[1-(4-Fluorophenoxymethyl)-1H-benzimidazol-2-yl]-furazan-3-ylamine,-   4-[1-(3,4-Dimethylphenoxymethyl)-1H-benzimidazol-2-yl]-furazan-3-yl-N-(2-cyanoethyl)-amine,    and compounds represented by the formula:

wherein R and R¹ are as defined below

R R¹

H

H

H

H

H

CH₂CH₂CN

CH₂CH₂CN

CH₂CH₂CN

H

H

H

H

H

H

H

H

CH₂CH₂CN

CH₂CH₂CH₂OH

H

Hor pharmaceutically acceptable derivatives thereof.

In still yet another preferred embodiment the compound of generalformula I according to the invention is:

wherein R, R⁴ and R⁵ are as defined below

R R⁴ R⁵

Me Me

Me Me

Me Me

Me Me

Me Me

OMe OMe

OMe OMe

OMe OMe

OMe OMe

OMe OMe

or pharmaceutically acceptable derivatives thereof.

More preferably, the compound according to the invention is a compoundof general formula I

whereinR represents phenyl or pyridinylwherein phenyl is optionally substituted by one or two substituentsindependently selected from lower alkyl, lower alkoxy, amino,acetylamino, halogen and nitro; and wherein pyridinyl is optionallysubstituted by amino or halogen;X represents a group C═O;R¹ represents hydrogen or cyano-lower alkyl;R², R³, R⁴, R⁵ and R⁶ represent hydrogen;and pharmaceutically acceptable derivatives thereof,and wherein the prefix lower denotes a radical having up to andincluding a maximum of 7, especially up to and including a maximum of 4carbon atoms.

Especially preferably, the compound according to the invention isrepresented by the following formula

wherein R, Y and R¹ are defined as follows:

R Y R¹

O H

O CH₂CH₂CN

O H

O CH₂CH₂CNor pharmaceutically acceptable derivatives thereof.

More especially preferably, the compound according to the invention isrepresented by the following formula

wherein R, Y and R1 are defined as follows:

R Y R1

O CH₂CH₂CN

O H

O CH₂CH₂CN

or pharmaceutically acceptable derivatives thereof.

Particularly preferably, the compound of general formula I according tothe invention is

or pharmaceutically acceptable derivatives thereof.

The term derivative or derivatives in the phrase “pharmaceuticallyacceptable derivative” or “pharmaceutically acceptable derivatives” ofcompounds of general formula I relates to salts, solvates and complexesthereof and to solvates and complexes of salts thereof, as well as topro-drugs, polymorphs, and isomers thereof (including optical, geometricand tautomeric isomers) and also salts of pro-drugs thereof. In a morepreferred embodiment, it relates to salts and pro-drugs, as well as tosalts of pro-drugs thereof.

Salts are preferably acid addition salts. Salts are formed, preferablywith organic or inorganic acids, from compounds of formula (I) with abasic nitrogen atom, especially the pharmaceutically acceptable salts.Suitable inorganic acids are, for example, halogen acids, such ashydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organicacids are, for example, carboxylic, phosphonic, sulfonic or sulfamicacids, for example acetic acid, propionic acid, octanoic acid, decanoicacid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid,succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid,malic acid, tartaric acid, citric acid, amino acids, such as glutamicacid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleicacid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoicacid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylaceticacid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid,1,5-naphthalene-disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid,methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid,N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamicacid, or other organic protonic acids, such as ascorbic acid.

The compound according to the invention may be administered in the formof a pro-drug which is broken down in the human or animal body to give acompound of the formula I. Examples of pro-drugs include in vivohydrolysable esters and amides of a compound of the formula I.Particular pro-drugs considered are ester and amides of naturallyoccurring amino acids and ester or amides of small peptides, inparticular small peptides consisting of up to five, preferably two orthree amino acids, as well as esters and amides of pegylated hydroxyacids, preferably hydroxy acetic acid and lactic acid. Pro-drug estersare formed from the acid function of the amino acid or the C terminal ofthe peptide and suitable hydroxy group(s) in the compound of formula I.Pro-drug amides are formed from the amino function of the amino acid orthe N terminal of the peptide and suitable carboxy group(s) in thecompound of formula I, or from the acid function of the amino acid orthe C terminal of the peptide and suitable amino group(s) in thecompound of formula I. Particularly preferably the pro-drug amides areformed from the amino group(s) present within the R group of formula I.

More preferably, the pro-drug is formed by the addition of glycine,alanine or lysine to the compound of formula I.

Even more preferably the compound of general formula I is in the form ofa pro-drug selected from the compounds of formulae:

In an especially preferred embodiment the compound according to theinvention is in the form of a pro-drug which has the following formula

In a most especially preferred embodiment the compound according to theinvention is a pharmaceutically acceptable salt, preferably ahydrochloride salt thereof, most preferably a dihydrochloride saltthereof, of a compound of the following formula

The pharmaceutically active metabolite in vivo in this case is BAL27862.

These pro-drugs may be prepared by processes that are known per se, inparticular, a process, wherein a compound of formula (II)

wherein R¹ is defined as for formula (I) and Z is CH or N, or aderivative of such a compound comprising functional groups in protectedform,or a salt thereof is(1) acylated with an amino acid of formula (III)

whereinR¹⁰ is selected from hydrogen (Gly); methyl (Ala) and protectedaminobutyl (Lys) and

R¹¹ is a suitable amino protecting group, and

(2) any protecting groups in a protected derivative of the resultingcompound are removed to yield a pro-drug as shown above, and, if sodesired,(3) said pro-drug is converted into a salt by treatment with an acid, ora salt of a compound of formula (II) is converted into the correspondingfree compound of formula (II) or into another salt, and/or a mixture ofisomeric product compounds is separated into the individual isomers.

Acylation of a compound of formula (II) with an amino acid of formula(III) is performed in a manner known per se, usually in the presence ofa suitable polar or dipolar aprotic solvent, with cooling or heating asrequired, for example in a temperature range from approximately minus80° C. to approximately plus 150° C., more preferably from minus 30° C.to plus 120° C., especially in a range from approximately around 0° C.to the reflux temperature of the used solvent. Optionally a suitablebase is added, in particularly an aromatic base like pyridine orcollidine or a tertiary amine base such as triethylamine ordiisopropylethylamine, or an inorganic basic salt, e.g. potassium orsodium carbonate.

Acylation may be accomplished under conditions used for amide formationknown per se in peptide chemistry, e.g. with activating agents for thecarboxy group, such as carbodiimides like N,N′-diethyl-, N,N′-dipropyl-,N,N′-diisopropyl-, N,N′-dicyclohexylcarbodiimide andN-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide-hydrochloride (EDC),or with agents such as 1-hydroxybenzotriazole (HOBt),benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate(BOP), O-(7-aza-benzotriazol-1-yl)-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HATU),2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TPTU), optionally in the presence of suitable bases, catalysts orco-reagents. The carboxy group may also be activated as acyl halogenide,preferably as acyl chloride, e.g. by reaction with thionylchloride oroxalylchloride, or as symmetrical or unsymmetrical anhydride, e.g. byreaction with halogeno formates like ethyl chloroformate, optionally inthe presence of suitable bases, catalysts or co-reagents.

If one or more other functional groups, for example carboxy, hydroxy oramino, are or need to be protected in a compound of formula (II) or(III), because they should not take part in the reaction, these are suchprotecting groups as are usually applied in the synthesis of amideslike, in particular peptide compounds, cephalosporins, penicillins,nucleic acid derivatives and sugars, which are known to the skilledpersons. Suitable protecting groups for amino groups are for examplet-butyl carbamate, benzyl carbamate or 9-fluorenylmethyl carbamate.

The protecting groups may already be present in precursors and shouldprotect the functional groups concerned against unwanted secondaryreactions, such as alkylations, acylations, etherifications,esterifications, oxidations, solvolysis, and similar reactions. It is acharacteristic of protecting groups that they lend themselves readily,i.e. without undesired secondary reactions, to removal, typically bysolvolysis, reduction, photolysis or also by enzyme activity, forexample under conditions analogous to physiological conditions, and thatthey are not present in the end products. The specialist knows, or caneasily establish, which protecting groups are suitable with thereactions mentioned hereinabove and hereinafter.

The protection of such functional groups by such protecting groups, theprotecting groups themselves, and their removal reactions are describedfor example in standard reference books for peptide synthesis and inspecial books on protective groups such as

-   J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum    Press, London and New York 1973, in “Methoden der organischen    Chemie” (Methods of organic chemistry), Houben-Weyl, 4th edition,    Volume 15/I, Georg Thieme Verlag, Stuttgart 1974, and in T. W.    Greene, G. M. Wuts “Protective Groups in Organic Synthesis”, Wiley,    New York, 2006.

Disease

The compounds of general formula I according to the invention have beenshown to arrest cell proliferation and induce cell death, for example byapoptosis.

Deregulation of cell proliferation, or lack of appropriate cell death,has wide ranging clinical implications. A number of diseases associatedwith such deregulation involve hyperproliferation, inflammation, tissueremodeling and repair. Familiar indications in this category includecancers, restenosis, neointimal hyperplasia, angiogenesis,endometriosis, lymphoproliferative disorders, transplantation relatedpathologies (graft rejection), polyposis, loss of neural function in thecase of tissue remodeling and the like.

Cancer is associated with abnormal cell proliferation and cell deathrates. As apoptosis is inhibited or delayed in most types ofproliferative, neoplastic diseases, induction of apoptosis is an optionfor treatment of cancer, especially in cancer types which showresistance to classic chemotherapy, radiation and immunotherapy(Apoptosis and Cancer Chemotherapy, Hickman and Dive, eds., BlackwellPublishing, 1999). Also in autoimmune and transplantation relateddiseases and pathologies compounds inducing apoptosis may be used torestore normal cell death processes and therefore can eradicate thesymptoms and might cure the diseases. Further applications of compoundsinducing apoptosis may be in restenosis, i.e. accumulation of vascularsmooth muscle cells in the walls of arteries, and in persistentinfections caused by a failure to eradicate bacteria- and virus-infectedcells. Furthermore, apoptosis can be induced or reestablished inepithelial cells, in endothelial cells, in muscle cells, and in otherswhich have lost contact with extracellular matrix.

A compound according to general formula I or pharmaceutically acceptablederivatives thereof may be used for the prophylactic or especiallytherapeutic treatment of the human or animal body, in particular fortreating a neoplastic disease, autoimmune disease, transplantationrelated pathology and/or degenerative disease. Examples of suchneoplastic diseases include, but are not limited to, epithelialneoplasms, squamous cell neoplasms, basal cell neoplasms, transitionalcell papillomas and carcinomas, adenomas and adenocarcinomas, adnexaland skin appendage neoplasms, mucoepidermoid neoplasms, cysticneoplasms, mucinous and serous neoplasms, ducal-, lobular and medullaryneoplasms, acinar cell neoplasms, complex epithelial neoplasms,specialized gonadal neoplasms, paragangliomas and glomus tumours, naeviand melanomas, soft tissue tumours and sarcomas, fibromatous neoplasms,myxomatous neoplasms, lipomatous neoplasms, myomatous neoplasms, complexmixed and stromal neoplasms, fibroepithelial neoplasms, synovial likeneoplasms, mesothelial neoplasms, germ cell neoplasms, trophoblasticneoplasms, mesonephromas, blood vessel tumours, lymphatic vesseltumours, osseous and chondromatous neoplasms, giant cell tumours,miscellaneous bone tumours, odontogenic tumours, gliomas,neuroepitheliomatous neoplasms, meningiomas, nerve sheath tumours,granular cell tumours and alveolar soft part sarcomas, Hodgkin's andnon-Hodgkin's lymphomas, other lymphoreticular neoplasms, plasma celltumours, mast cell tumours, immunoproliferative diseases, leukemias,miscellaneous myeloproliferative disorders, lymphoproliferativedisorders and myelodysplastic syndromes.

The compounds of general formula I or pharmaceutically acceptablederivatives thereof may be used to treat autoimmune diseases. Examplesof such autoimmune diseases include, but are not limited to, systemic,discoid or subacute cutaneous lupus erythematosus, rheumatoid arthritis,antiphospholipid syndrome, CREST, progressive systemic sclerosis, mixedconnective tissue disease (Sharp syndrome), Reiter's syndrome, juvenilearthritis, cold agglutinin disease, essential mixed cryoglobulinemia,rheumatic fever, ankylosing spondylitis, chronic polyarthritis,myasthenia gravis, multiple sclerosis, chronic inflammatorydemyelinating polyneuropathy, Guillan-Barre syndrome,dermatomyositis/polymyositis, autoimmune hemolytic anemia,thrompocytopenic purpura, neutropenia, type I diabetes mellitus,thyroiditis (including Hashimoto's and Grave' disease), Addison'sdisease, polyglandular syndrome, pemphigus (vulgaris, foliaceus,sebaceous and vegetans), bullous and cicatricial pemphigoid, pemphigoidgestationis, epidermolysis bullosa acquisita, linear IgA disease, lichensclerosus et atrophicus, morbus Duhring, psoriasis vulgaris, guttate,generalized pustular and localized pustular psoriasis, vitiligo,alopecia greata, primary biliary cirrhosis, autoimmune hepatitis, allforms of glomerulonephritis, pulmonal hemorrhage (goodpasture syndrome),IgA nephropathy, pernicious anemia and autoimmune gastritis,inflammatory bowel diseases (including colitis ulcerosa and morbusCrohn), Behcet's disease, Celic-Sprue disease, autoimmune uveitis,autoimmune myocarditis, granulomatous orchitis, aspermatogenesis withoutorchitis, idiopatic and secondary pulmonary fibrosis, inflammatorydiseases with a possibility of autoimmune pathogenesis, such as pyodermagangrensosum, lichen ruber, sarcoidosis (including Lofgren andcutaneous/subcutaneous type), granuloma anulare, allergic type I andtype IV immunolgical reaction, asthma bronchiale, pollinosis, atopic,contact and airborne dermatitis, large vessel vasculitis (giant cell andTakayasu's arteritis), medium sized vessel vasculitis (polyarteritisnodosa, Kawasaki disease), small vessel vasculitis (Wegener'sgranulomatosis, Churg Strauss syndrome, microscopic polangiitis,HenochSchoenlein purpura, essential cryoglobulinemic vasculitis,cutaneous leukoklastic angiitis), hypersensitivity syndromes, toxicepidermal necrolysis (Stevens-Johnson syndrome, erythema multiforme),diseases due to drug side effects, all forms of cutaneous,organ-specific and systemic effects due to type I-vu (Coombsclassification) immunologic forms of reaction, transplantation relatedpathologies, such as acute and chronic graft versus host and host versusgraft disease, involving all organs (skin, heart, kidney, bone marrow,eye, liver, spleen, lung, muscle, central and peripheral nerve system,connective tissue, bone, blood and lymphatic vessel, genito-urinarysystem, ear, cartillage, primary and secondary lymphatic systemincluding bone marrow, lymph node, thymus, gastrointestinal tract,including oro-pharynx, esophageus, stomach, small intestine, colon, andrectum, including parts of above mentioned organs down to single celllevel and substructures, e.g. stem cells).

Particularly preferably, the disease according to the invention is aneoplastic or autoimmune disease. In an especially preferred embodimentthe disease is cancer.

Examples of cancers in terms of the organs and parts of the bodyaffected include, but are not limited to, the breast, cervix, ovaries,colon, rectum, (including colon and rectum i.e. colorectal cancer),lung, (including small cell lung cancer, non-small cell lung cancer,large cell lung cancer and mesothelioma), endocrine system, bone,adrenal gland, thymus, liver, stomach, intestine, (including gastriccancer), pancreas, bone marrow, hematological malignancies, (such aslymphoma, leukemia, myeloma or lymphoid malignancies), bladder, urinarytract, kidneys, skin, thyroid, brain, head, neck, prostate and testis.Preferably the cancer is selected from the group consisting of breastcancer, prostate cancer, cervical cancer, ovarian cancer, gastriccancer, colorectal cancer, pancreatic cancer, liver cancer, braincancer, neuroendocrine cancer, lung cancer, kidney cancer, hematologicalmalignancies, melanoma and sarcomas. Especially preferably the cancer isselected from the group consisting of breast cancer, cervical cancer,gastric cancer, lung cancer, colorectal cancer and melanoma. Moreespecially preferably the cancer is selected from the group consistingof gastric cancer, lung cancer, colorectal cancer and melanoma.

Samples

The measurement of the level of phospho-Akt may be performed in vitro,on a sample of biological material derived from the subject. The samplemay be any biological material separated from the body such as, forexample, normal tissue, tumour tissue, cell lines, plasma, serum, wholeblood, cerebrospinal fluid, lymph fluid, circulating tumour cells, celllysate, tissue lysate, urine and aspirates. Preferably the sample isderived from the group consisting of normal tissue, tumour tissue, celllines and circulating tumour cells. More preferably the sample isderived from tumour tissue or circulating tumour cells. In oneparticularly preferred embodiment the sample is derived from tumourtissue. For example, the level of phospho-Akt may be measured in afresh, frozen or formalin fixed/paraffin embedded tumour tissue sample.

The sample is pre-obtained from the subject before the sample issubjected to the method steps involving measuring the level of thebiomarker. The methods for removal of the sample are well known in theart, and it may for example be removed from the subject by biopsy, forexample by punch biopsy, core biopsy, aspiration fine needle biopsy,endoscopic biopsy, or surface biopsy. A whole blood, plasma or serumsample may be collected by venipuncture and further processed accordingto standard techniques. Circulating tumour cells may also be obtainedfrom blood based on, for example, size (e.g. ISET—Isolation by Size ofEpithelial Tumour cells) or immunomagnetic cell enrichment (e.g.CellSearch®, Veridex, Raritan, N.J.).

Sample Comparison

The subject according to the invention may be human or animal.Preferably the subject is human.

The biomarker phospho-Akt is measured ex vivo in a sample or samplestaken from the human or animal body, preferably taken from the humanbody. The sample or samples are pre-obtained from the human or animalbody, preferably pre-obtained from the human body before the sample issubjected to the method steps involving measuring the level of thebiomarker.

A biomarker is in general a substance that is used as an indicator of abiological response, preferably as an indicator of the susceptibility toa given treatment, which in the present application is treatment with acompound of general formula I or pharmaceutically acceptable derivativesthereof.

In a particularly preferred embodiment, higher phospho-Akt levels in thesample relative to a standard value or set of standard values predictsresistance.

The higher phospho-Akt levels may arise due to higher total Akt levelsin the sample and/or a higher percentage of Akt which becomesphosphorylated.

As used herein, an increase or relatively high or high or higher levelsrelative to a standard level or set of standard levels means the amountor concentration of the biomarker in a sample is detectably greater inthe sample relative to the standard level or set of standard levels.This encompasses at least an increase of, or higher level of, about 1%relative to the standard, preferably at least an increase of about 5%relative to the standard. More preferably it is an increase of, orhigher level of, at least about 10% relative to the standard. Moreparticularly preferably it is an increase of, or higher level of, atleast about 20% relative to the standard. For example, such an increaseof, or higher level of, may include, but is not limited to, at leastabout 1%, about 10%, about 20%, about 30%, about 50%, about 70%, about80%, about 100%, about 150% or about 200% or more relative to thestandard.

Preferably, higher phospho-Akt levels in a sample or samples

i) relative to a standard value or set of standard values from subjectswith the same tumour histotype; or

ii) relative to a standard value or set of standard values from normalcells, tissue or body fluid;

are predictive of resistance.

The measuring of a level of phospho-Akt is performed ex-vivo in a samplepre-obtained from the subject.

Especially preferably, higher phospho-Akt levels in a sample or samplesrelative to a standard value or set of standard values from subjectswith the same tumour histotype are predictive of resistance.

In one preferred embodiment, for the case i) where the measurement iscompared in a sample or samples relative to a standard value or set ofstandard values from samples from subjects with the same tumourhistotype as the sample to which it is to be compared, the standardvalue or set of standard values are established from samples from apopulation of subjects with that cancer type. The samples from thesestandard subjects may for example be derived from the tumour tissue orcirculating tumour cells, as long as the origin of the sample isconsistent between the standard and the sample to be compared.

In another preferred embodiment, for the case ii) where the measurementis compared in a sample or samples relative to a standard value or setof standard values taken from normal cells or tissue, the standard valueor set of standard values may be established from a sample of normal(e.g. non-tumorous) cells, tissue or body fluid. Such data may begathered from a population of subjects in order to develop the standardvalue or set of standard values.

The standard value or set of standard values are established ex-vivofrom pre-obtained samples which may be from cell lines, or preferablybiological material taken from at least one subject and more preferablyfrom an average of subjects (e.g., n=2 to 1000 or more). The standardvalue or set of standard values may then be correlated with the responsedata of the same cell lines, or same subjects, to treatment with acompound of general formula I or a pharmaceutically acceptablederivative thereof. From this correlation a comparator module, forexample in the form of a relative scale or scoring system, optionallyincluding cut-off or threshold values, can be established whichindicates the levels of biomarker associated with a spectrum of responselevels to the compound of formula I or a pharmaceutically acceptablederivative thereof. The spectrum of response levels may compriserelative sensitivity to the therapeutic activity of the compound, (e.g.high sensitivity to low sensitivity), as well as resistance to thetherapeutic activity. In a preferred embodiment this comparator modulecomprises a cut-off value or set of values which predicts resistance totreatment.

For example, if an immunohistochemical method is used to measure thelevel of phospho-Akt in a sample, standard values may be in the form ofa scoring system. Such a system might take into account the percentageof cells in which staining for phospho-Akt is present. The system mayalso take into account the relative intensity of staining or cellularlocalisation in the individual cells. The standard values or set ofstandard values of the level of phospho-Akt may then be correlated withdata indicating the response, especially resistance, of the subject ortissue or cell line to the therapeutic activity of a compound of formulaI or a pharmaceutically acceptable derivative thereof. Such data maythen form part of a comparator module.

Response is the reaction of the cell lines, or preferably of thesubject, or more preferably of the disease in a subject, to thetherapeutic activity of a compound of general formula I or apharmaceutically acceptable derivative thereof. The spectrum of responselevels may comprise relative sensitivity to the therapeutic activity ofthe compound, (e.g. high sensitivity to low sensitivity), as well asresistance to the therapeutic activity. The response data may forexample be monitored in terms of: objective response rates, time todisease progression, progression free survival, and overall survival.

The response of a cancerous disease may be evaluated by using criteriawell known to a person in the field of cancer treatment, for example butnot restricted to:

-   Response Evaluation Criteria in Solid Tumors (RECIST) Guidelines,    Source: Eisenhauer E A, Therasse P, Bogaerts J, Schwartz L H,    Sargent D, Ford R, Dancey J, Arbuck S Gwyther S, Mooney M,    Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J. New    response evaluation criteria in solid tumours: revised RECIST    guideline (version 1.1). Eur J. Cancer. 2009; 45:228-47;-   RANO Criteria for High-Grade Gliomas, Source: Wen P Y, Macdonald D    R, Reardon D A, Cloughesy T F, Sorensen A G, Galanis E, Degroot J,    Wick W, Gilbert M R, Lassman A B, Tsien C, Mikkelsen T, Wong E T,    Chamberlain M C, Stupp R, Lamborn K R, Vogelbaum M A, van den Bent M    J, Chang S M. Updated response assessment criteria for high-grade    gliomas: response assessment in neuro-oncology working group. J Clin    Oncol. 2010; 28(11):1963-72;-   CA-125 Rustin Criteria for Ovarian Cancer Response, Source: Rustin G    J, Quinn M, Thigpen T, du Bois A, Pujade-Lauraine E, Jakobsen A,    Eisenhauer E, Sagae S, Greven K, Vergote I, Cervantes A,    Vermorken J. Re: New guidelines to evaluate the response to    treatment in solid tumors (ovarian cancer). J Natl Cancer Inst.    2004; 96(6):487-8;    and-   PSA Working Group 2 Criteria for Prostate Cancer Response, Source:    Scher H I, Halabi S, Tannock I, Morris M, Sternberg C N, Carducci M    A, Eisenberger M A, Higano C, Bubley G J, Dreicer R, Petrylak D,    Kantoff P, Basch E, Kelly W K, Figg W D, Small E J, Beer T M,    Wilding G, Martin A, Hussain M; Prostate Cancer Clinical Trials    Working Group. Design and end points of clinical trials for patients    with progressive prostate cancer and castrate levels of    testosterone: recommendations of the Prostate Cancer Clinical Trials    Working Group. J Clin Oncol. 2008; 26(7):1148-59.

Resistance is associated with there not being an observable and/ormeasurable reduction in, or absence of, one or more of the following:reduction in the number of abnormal cells, preferably cancerous cells,or absence of the abnormal cells, preferably cancerous cells; forcancerous diseases: reduction in tumour size; inhibition (i.e., slowedto some extent and preferably stopped) of further tumour growth;reduction in the levels of tumour markers such as PSA and CA-125;inhibition (i.e., slowed to some extent and preferably stopped) ofcancer cell infiltration into other organs (including the spread ofcancer into soft tissue and bone); inhibition (i.e., slowed to someextent and preferably stopped) of tumour metastasis; alleviation of oneor more of the symptoms associated with the specific cancer; and reducedmorbidity and mortality.

In a preferred embodiment resistance means there is no observable and/ormeasurable reduction in, or absence of, one or more of the followingcriteria: reduction in tumour size; inhibition of further tumour growth;inhibition of cancer cell infiltration into other organs; and inhibitionof tumour metastasis.

In a more preferred embodiment resistance refers to one or more of thefollowing criteria: no reduction in tumour size; no inhibition offurther tumour growth, no inhibition of cancer cell infiltration intoother organs; and no inhibition of tumour metastasis.

Measurement of the aforementioned resistance criteria is according toclinical guidelines well known to a person in the field of cancertreatment, such as those listed above for measuring the response of acancerous disease.

Response may also be established in vitro by assessing cellproliferation and/or cell death. For example, effects on cell death orproliferation may be assessed in vitro by one or more of the followingwell established assays: A) Nuclear staining with Hoechst 33342 dyeproviding information about nuclear morphology and DNA fragmentationwhich are hallmarks of apoptosis. B) Annexin V binding assay whichreflects the phosphatidylserine content of the outer lipid bilayer ofthe plasma membrane. This event is considered an early hallmark ofapoptosis. C) TUNEL assay (Terminal deoxynucleotidyl transferasemediated dUTP Nick End Labeling assay), a fluorescence method forevaluating cells undergoing apoptosis or necrosis by measuring DNAfragmentation by labeling the terminal end of nucleic acids. D) MTSproliferation assay measuring the metabolic activity of cells. Viablecells are metabolically active whereas cells with a compromisedrespiratory chain show a reduced activity in this test. E) Crystalviolet staining assay, where effects on cell number are monitoredthrough direct staining of cellular components. F) Proliferation assaymonitoring DNA synthesis through incorporation of bromodeoxyuridine(BrdU). Inhibitory effects on growth/proliferation can be directlydetermined. G) YO-PRO assay which involves a membrane impermeable,fluorescent, monomeric cyanine, nucleic acid stain, which permitsanalysis of dying (e.g. apoptotic) cells without interfering with cellviability. Overall effects on cell number can also be analysed aftercell permeabilisation. H) Propidium iodide staining for cell cycledistribution which shows alterations in distribution among the differentphases of the cell cycle. Cell cycle arresting points can be determined.I) Anchorage-independent growth assays, such as colony outgrowth assayswhich assess the ability of single cell suspensions to grow intocolonies in soft agar.

In a preferred embodiment relating to determination of resistance invitro, resistance means there is no decrease in the proliferation rateof abnormal cells and/or reduction in the number of abnormal cells. Morepreferably resistance means there is no decrease in the proliferationrate of cancerous cells and/or no reduction in the number of cancerouscells. The reduction in the number of abnormal, preferably cancerous,cells may occur through a variety of programmed and non-programmed celldeath mechanisms. Apoptosis, caspase-independent programmed cell deathand autophagic cell death are examples of programmed cell death. Howeverthe cell death criteria involved in embodiments of the invention are notto be taken as limited to any one cell death mechanism.

phospho-Akt

As defined above, the term Akt is used herein to encompass all thepreviously mentioned synonyms and isoforms and phospho-Akt is Akt thathas been phosphorylated on one or more residues, with the proviso thatfor Akt1, Akt2, and Akt3 the designation phospho-Akt is used to indicatephosphorylation at a site other than T308, T309 or T305 respectively. Toclarify this further, the phospho-Akt may or may not be phosphorylatedat T308, T309 or T305 for Akt 1, Akt 2 and Akt3 respectively, but thedesignation phospho-Akt indicates phosphorylation at sites other thanthese.

Preferred examples of the protein sequence of Akt (human Akt) are listedin SEQ. ID No. 1 to 3, FIGS. 9-11. However the term Akt also encompasseshomologues, mutant forms, allelic variants, isoforms, splice variantsand equivalents of these sequences. The human homologues, mutant forms,allelic variants, isoforms, splice variants and equivalents of thesesequences are more preferred embodiments. More preferably it encompassessequences having at least about 75% identity, especially preferably atleast about 85% identity, particularly preferably at least about 95%identity, to any of the sequences represented by SEQ. ID. No. 1 to 3. Inan especially preferred embodiment, Akt corresponds to any of thesequences represented by SEQ ID NO. 1 to 3 and sequences having at least99% identity with any of these sequences. In a particularly preferredembodiment, Akt corresponds to the sequence represented by SEQ ID NO. 1and sequences having at least 95% identity with this sequence,preferably at least 99% identity. In a more particularly preferredembodiment, Akt corresponds to a sequence represented by any of SEQ IDNO. 1 to 3. In a still more particularly preferred embodiment Aktcorresponds to a sequence represented by SEQ ID NO. 1.

In a particularly preferred embodiment, phospho-Akt shall refer to Akt(wherein the preferred embodiments thereof are as given in the precedingparagraph) that has been phosphorylated on the following serine residue:

for Akt1 (SEQ. ID. No. 1): S473; for Akt2 (SEQ. ID. No. 2): S474; andfor Akt3 (SEQ. ID. No. 3): S472.

In yet another especially preferred embodiment, phospho-Akt correspondsto any of the sequences represented by SEQ ID NO. 1 to 3 and sequenceshaving at least 99% identity with any of these sequences, and whereinfor the sequence represented by SEQ. ID. No. 1, S473 is phosphorylated,or for the sequence represented by SEQ. ID. No. 2, S474 isphosphorylated, or for the sequence represented by SEQ. ID. No. 3, S472is phosphorylated.

In a very particularly preferred embodiment phospho-Akt corresponds to asequence represented by SEQ ID NO. 1 that has been phosphorylated onS473.

Level of Phospho-Akt

The level of phospho-Akt may be assayed in the sample by technical meanswell known to a skilled person. Examples of methods of proteinexpression analysis known in the art which are suitable to measure thelevel of phospho-Akt at the protein level include, but are not limitedto, i) immunohistochemistry (IHC) analysis, ii) western blotting iii)immunoprecipitation iv) enzyme linked immunosorbant assay (ELISA), v)radioimmunoassay, vi) Fluorescence activated cell sorting (FACS), vii)mass spectrometry, including matrix assisted laser desorption/ionization(MALDI, e.g. MALDI-MS) or electrospray (e.g. ESI-MS).

The antibodies involved in some of the above methods may be monoclonalor polyclonal antibodies, antibody fragments, and/or various types ofsynthetic antibodies, including chimeric antibodies. The antibody may belabeled to enable it to be detected or capable of detection followingreaction with one or more further species, for example using a secondaryantibody that is labeled or capable of producing a detectable result.Antibodies specific to phospho-Akt are available commercially from CellSignaling or can be prepared via conventional antibody generationmethods well known to a skilled person.

Preferred methods of protein analysis are ELISA, mass spectrometrytechniques, immunohistochemistry and western blotting, more preferablyELISA, western blotting and immunohistochemistry, particularlypreferably western blotting and immunohistochemistry. In westernblotting, also known as immunoblotting, labelled antibodies may be usedto assess levels of protein, where the intensity of the signal from thedetectable label corresponds to the amount of protein, and can bequantified for example by densitometry.

Immunohistochemistry again uses labelled antibodies to detect thepresence and relative amount of the biomarker. It can be used to assessthe percentage of cells for which the biomarker is present. It can alsobe used to assess the localisation or relative amount of the biomarkerin individual cells; the latter is seen as a function of the intensityof staining.

ELISA stands for enzyme linked immunosorbant assay, since it uses anenzyme linked to an antibody or antigen for the detection of a specificprotein. ELISA is typically performed as follows (although othervariations in methodology exist): a solid substrate such as a 96 wellplate is coated with a primary antibody, which recognises the biomarker.The bound biomarker is then recognised by a secondary antibody specificfor the biomarker. This may be directly joined to an enzyme or a thirdanti-immunoglobulin antibody may be used which is joined to an enzyme. Asubstrate is added and the enzyme catalyses a reaction, yielding aspecific colour. By measuring the optical density of this colour, thepresence and amount of the biomarker can be determined.

Uses of Biomarker

The biomarker may be used to predict inherent resistance of the diseasein a subject to the compound of general formula I or a pharmaceuticallyacceptable derivative thereof as defined above.

The biomarker may be used to select subjects suffering or predisposed tosuffering from a disease, preferably cancer, for treatment with acompound of general formula I or a pharmaceutically acceptablederivative thereof as defined above. The levels of such a biomarker maybe used to identify subjects likely to respond or to not respond totreatment with such agents. Stratification of subjects may be made inorder to avoid unnecessary treatment regimes. In particular thebiomarker may be used to identify subjects from whom a sample or samplesdo not display a higher level of phospho-Akt, relative to a standardlevel or set of standard levels, whereupon such subjects may then beselected for treatment with the compound of formula I or apharmaceutically acceptable derivative thereof as defined above.

The biomarker may also be used to assist in the determination oftreatment regimes, regarding amounts and schedules of dosing.Additionally, the biomarker may be used to assist in the selection of acombination of drugs to be given to a subject, including a compound orcompounds of general formula I or a pharmaceutically acceptablederivative thereof, and another chemotherapeutic (cytotoxic) agent oragents. Furthermore, the biomarker may be used to assist in thedetermination of therapy strategies in a subject including whether acompound of general formula I or a pharmaceutically acceptablederivative thereof is to be administered in combination with targetedtherapy, endocrine therapy, radiotherapy, immunotherapy or surgicalintervention, or a combination of these.

Phospho-Akt may also be used in combination with other biomarkers topredict the response to a compound of general formula I or apharmaceutically acceptable derivative thereof and to determinetreatment regimes. It may furthermore be used in combination withchemo-sensitivity testing to predict resistance and to determinetreatment regimes. Chemo-sensitivity testing involves directly applyinga compound of general formula I to cells taken from the subject, forexample from a subject with haematological malignancies or accessiblesolid tumours, for example breast, head and neck cancers or melanomas,to determine the response of the cells to the compound.

Method of Treatment

The invention also involves in some aspects a method of treatment andphospho-Akt for use in a method of treatment, wherein the level ofphospho-Akt is first established relative to a standard level or set ofstandard levels and then a compound of general formula I or apharmaceutically acceptable derivative thereof as defined above, isadministered if the level of phospho-Akt in said sample is not higherthan a standard value or set of standard values. The compound of formulaI or a pharmaceutically acceptable derivative thereof may beadministered in a pharmaceutical composition, as is well known to aperson skilled in the art. Suitable compositions and dosages are forexample disclosed in WO 2004/103994 A1 pages 35-39, which arespecifically incorporated by reference herein. Compositions for enteraladministration, such as nasal, buccal, rectal or, especially, oraladministration, and for parenteral administration, such as intravenous,intramuscular or subcutaneous administration, to warm-blooded animals,especially humans, are especially preferred. More particularly,compositions for intravenous administration are preferred.

The compositions comprise the active ingredient and a pharmaceuticallyacceptable carrier. An example of a composition includes, but is notlimited to, the following: 5000 soft gelatin capsules, each comprisingas active ingredient 0.05 g of one of the compounds of general formula(I), are prepared as follows: 250 g pulverized active ingredient issuspended in 2 liter Lauroglykol® (propylene glycol laurate, GattefosséS.A., Saint Priest, France) and ground in a wet pulverizer to produce aparticle size of about 1 to 3 μm. 0.419 g portions of the mixture arethen introduced into soft gelatin capsules using a capsule-fillingmachine.

The invention also relates in one aspect to a method of treating aneoplastic or autoimmune disease, preferably cancer, by first decreasingthe level of phospho-Akt in a subject that has a sample with a higherlevel of phospho-Akt compared to a standard level or set of standardlevels, then treating the subject with a compound of general formula Ior a pharmaceutically acceptable derivative as defined above. The levelof phospho-Akt may be decreased by direct or indirect chemical orgenetic means. Examples of such methods are treatment with a drug thatresults in reduced phospho-Akt levels, targeted delivery of viral,plasmid or peptide constructs or antibody or siRNA or antisense todownregulate the level of phospho-Akt. For example siRNA may be used toreduce the level of rictor expressed, thus reducing mTORC2 complexformation and activity, and thereby indirectly lower the level ofphosphorylated Akt. The subject may then be treated with a compound ofgeneral formula I or a pharmaceutically acceptable derivative thereof.

A compound of general formula I or a pharmaceutically acceptablederivative thereof can be administered alone or in combination with oneor more other therapeutic agents. Possible combination therapy may takethe form of fixed combinations, or the administration of a compound ofthe invention and one or more other therapeutic agents which arestaggered or given independently of one another, or the combinedadministration of fixed combinations and one or more other therapeuticagents. A compound of general formula I or a pharmaceutically acceptablederivative thereof can, besides or in addition, be administeredespecially for tumour therapy in combination with chemotherapy(cytotoxic), targeted therapy, endocrine therapy, radiotherapy,immunotherapy, surgical intervention, or a combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above. Otherpossible treatments are therapy to maintain the patient's status aftertumour regression, or even chemo-preventive therapy, for example inpatients at risk.

Kit and Device

In one aspect the invention relates to a kit, and in another aspect to adevice, for predicting the response, preferably of a disease in asubject, to a compound of general formula I or a pharmaceuticallyacceptable derivative thereof as defined above, comprising reagentsnecessary for measuring the level of phospho-Akt in a sample.Preferably, the reagents comprise a capture reagent comprising adetector for phospho-Akt and a detector reagent.

The kit and device may also preferably comprise a comparator modulewhich comprises a standard value or set of standard values to which thelevel of phospho-Akt in the sample is compared. In a preferredembodiment, the comparator module is included in instructions for use ofthe kit. In another preferred embodiment the comparator module is in theform of a display device, for example a strip of colour or numericallycoded material which is designed to be placed next to the readout of thesample measurement to indicate resistance levels. The standard value orset of standard values may be determined as described above.

The reagents are preferably antibodies or antibody fragments whichselectively bind to phospho-Akt. These may for example be in the form ofone specific primary antibody which binds to phospho-Akt and a secondaryantibody which binds to the primary antibody, and which is itselflabelled for detection. The primary antibody may also be labelled fordirect detection. The kits or devices may optionally also contain a washsolution(s) that selectively allows retention of the bound biomarker tothe capture reagent as compared with other biomarkers after washing.Such kits can then be used in ELISA, western blotting, flow cytometry,immunohistochemical or other immunochemical methods to detect the levelof the biomarker.

Furthermore the device may comprise imaging devices or measurementdevices (for example, but not restricted to, measurement offluorescence) which further process the measured signals and transferthem into a scale in a comparator module.

More preferably the kit comprises a compound of general formula I, or apharmaceutically acceptable derivative thereof as defined above. Thiscompound may then be administered to the subject, in accordance with thelevel of the biomarker in the sample from the subject, as measured bythe reagents comprised in the kit. Therefore the kit according to theinvention may be used in the method of treatment according to theinvention, as defined above. In an especially preferred embodiment thekit comprises a compound of the following formula or a pharmaceuticallyacceptable salt thereof

In a particularly preferred embodiment of the kit the pharmaceuticallyacceptable salt is a dihydrochloride salt. In another aspect theinvention relates to the use of such a kit as described above.

In the present specification the words “comprise” or “comprises” or“comprising” are to be understood as to imply the inclusion of a stateditem or group of items, but not the exclusion of any other item or groupof items.

Experimental Methodology

Immunofluorescent Staining of Cultured Cells

A549 human non-small cell lung cancer (NSCLC, ATCC reference numberCCL-185) cells, HeLa cervical cancer cells (ATCC reference number CCL-2)and SKBR3 breast carcinoma cells (ATCC reference number HTB-30) wereseeded at densities of 50% on round microscope coverslips and culturedfor 24 hours in RPMI-1640 containing 10% FCS (also referred to as FBS)at 37° C., 5% CO₂. Compounds to be tested were dissolved in DMSO. Thecell culture medium was replaced with medium containing the dilutedcompound(s) (paclitaxel, vinblastine, colchicine and nocodazole werepurchased from Sigma-Aldrich) or vehicle. After treatment for the timesindicated in the Brief Description of the Figures, coverslips werewashed and cells were fixed in methanol/acetone (1:1) for 5 minutes atroom temperature and subsequently incubated in blocking buffer (0.5% BSAand 0.1% TX-100 in PBS) for 30 minutes at room temperature. Specimenswere then incubated with anti-alpha-tubulin antibody (Sigma, 1:2000) for1 hour at room temperature in blocking buffer. After several washingsteps cells were incubated with AlexaFluor-488 goat-anti-mouse IgG(Molecular Probes, 1:3000) for 1 hour at room temperature followed byseveral washing steps with blocking buffer. Specimens were then mountedwith ProLong Gold antifade (Molecular Probes), sealed with nail polishand examined with a Leica immunofluorescence microscope. Images werecaptured with a cooled CCD-camera and processed by ImageJ software.

Colony Outgrowth Assay:

Single cell suspensions of patient-derived tumour xenografts (maintainedin nude mice) were prepared. For colony outgrowth assays, cells wereplated in soft agar in 24-well plates according to the assay introducedby Hamburger & Salmon (Primary bioassay of human tumour stem cells,Science, 1977, 197:461-463). 2×10⁴-6×10⁴ cells in 0.2 mL mediumcontaining 0.4% agar were plated out on a bottom layer of 0.75% agar.Test compounds were applied in 0.2 mL culture medium. Every 24-wellplate contained untreated controls and samples in triplicates. Cultureswere incubated at 37° C. and 7.5% CO₂ for 5-28 days. 24 hours prior toanalysis, vital colonies were stained with a solution of metabolizabletetrazolium salt (Alley M C et al, Life Sci. 1982, 31:3071-3078) andwere counted with an automatic image analysis system (Omnicon 3600,Biosys GmbH).

Relative drug effects were expressed by the ratio of the mean number ofcolonies in the treated wells and the control wells. IC₇₀-values weredetermined by plotting compound concentrations versus relative colonycounts.

Protein Extraction

Tumours were extracted in ice-cold buffer containing 50 mM HEPES (pH7.5), 150 mM NaCl, 25 mM β-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mMEDTA, 0.1% NP40, 15 mM pyrophosphate, 2 mM sodium orthovanadate, 10 mMsodium molybdate, leupeptin (10 μg/mL), aprotinin (10 μg/mL) and 1 mMPMSF (1 mL extraction volume per 45 mg tumour). After homogenisation byPolytron, lysates were adjusted to 1% NP40 and incubated on ice for 20min. Lysates were clarified by centrifugation and frozen at −80° C.

Immunoblotting/Western Blotting

Immunoblotting was performed using 20 μg of total protein per lane.Protein concentration was determined with the BCA Protein Assay(Pierce). Protein was separated on a 10% SDS-gel and transferred to aPVDF membrane using Wet Blotting (45 min, 250 mA/gel). The primaryantibodies used for immunoblotting were as follows:

Phospho-Akt (serine 473) (available from Cell Signalling, referencenumber 9271) origin: rabbit polyclonal antibody, dilution 1:1000, bufferconditions: PBS containing 0.5% milk/0.1% tween;Akt protein (available from Epitomics, reference number 1085-1) origin:rabbit monoclonal antibody, dilution 1:2000, buffer conditions: PBScontaining 0.5% milk/0.1% tween;Actin: (available from Chemicon, reference number MAB1501) origin:mouse, monoclonal, dilution 1:5000, buffer conditions: PBS containing0.5% milk/0.1% tween.

The secondary antibodies used for immunoblotting wereperoxidase-conjugated goat anti-rabbit or goat anti-mouse (availablefrom Jackson ImmunoResearch Laboratories INC: reference number111-035-144 JIR and 115-035-146 JIR), dilution 1:5000, bufferconditions: 0.5% milk in PBS/0.1° A Tween. Labelled bands were revealedusing a Raytest Stella 3200 High Performance Imaging System.

Immunohistochemistry

Fixation of patient-derived tumour xenografts (maintained in nude mice)was performed in 10% neutral-buffered formalin containing 4%formaldehyde for 20-28 hours at room temperature. Fixed specimens werekept in a solution of 70% ethanol for a maximum of one week prior todehydration and paraffin embedding according to a standard procedure,using the conditions listed below:

Sequential Treatment time (hours) 70% EtOH 1 80% EtOH 2 99% EtOH 1 100%Isopropanol 0.5 100% Isopropanol 1 Xylol 0.5 Xylol 1 Xylol 1 Paraffin 1Paraffin 2 Paraffin 2

Paraffin sections of approximately 2 μm were cut and processed by usingthe automated immunostainer Benchmark XT® (Roche) running the standardprocessing steps. The visualisation of the specific antibody stainingwas done with DAB (3,3-diaminobenzidine) as chromogenic substrate at aconcentration of 5 mg/mL. The following primary antibody and processingconditions were used for staining:

Antibody Specification Processing Anti-Akt-pS473 from Dako, MTec100/30:EDTA-citrate buffer # M3628 rabbit monoclonal retrieval pH 8, 100° C.for 30 minutes. antibody Antibody incubation at 37° C. for 32 minutes ata dilution of 1:20

DETAILED EXAMPLES Example 1 A Distinct Mitotic Phenotype Induced byCompounds of General Formula I

Treatment with compound A (BAL27862) or with compound B, or compound Cinduced a highly reproducible and distinct microtubule phenotype in alltumour cell lines tested (shown for BAL27862 in A549, HeLa and SKBR3cells in FIG. 1, and for compound C and compound B in A549 cells in FIG.2). In dividing cells an apparent fragmentation of the mitotic spindleoccurred, resulting in the formation of dot-like structures (FIG. 1).This phenotype was shown to be distinct from that observed withconventional microtubule targeting agents, such as the microtubulestabiliser paclitaxel and the microtubule destabilisers vinblastine andcolchicine (FIG. 3) and nocodazole (FIG. 4).

Example 2 BAL27862 Overcomes Microtubule Phenotype Induced byConventional Microtubule-Targeting Drugs in a Dominant Fashion

In order to show the uniqueness of its activity on microtubules,BAL27862 was tested in combination with vinblastine, colchicine andpaclitaxel (FIG. 5) and nocodazole (FIG. 6) using A549 cells. Treatmentwith vinblastine, colchicine, paclitaxel or nocodazole alone induced themitotic microtubule phenotypes characteristic of these agents. However,combination treatment with BAL27862 for the last 4 hours resulted indisruption of the microtubule structures; creating a phenotypeconsistent with treatment of BAL27862 alone, despite the continuedpresence of vinblastine, colchicine, paclitaxel or nocodazole. Incontrast, treating first with BAL27862 and subsequently for 4 hours incombination with vinblastine, colchicine, paclitaxel or nocodazole hadno impact on the observed microtubule phenotype that was consistent withtreatment with BAL27862.

These data demonstrate that compounds of formula I affect microtubulebiology consistently, but in a different manner than conventionalmicrotubule targeting agents.

Detailed Examples According to the Invention Example 3 Association ofHigh Phospho-Akt Expression Levels with Patient-Derived Tumour CellsResistant to BAL27862 Treatment

Based on colony outgrowth assays, using tumour cells derived from 8patient-derived tumours maintained as xenografts in mice,BAL27862-sensitive or resistant tumour cells were identified fromgastric cancer, colorectal cancer, melanoma, and lung cancer (see Table1). Concentrations at which 70% growth inhibition was observed versuscontrols (IC₇₀) are shown in Table 1. In this table, BAL27862-sensitivetumour cells were those that had IC₇₀ values in the low nanomolar range,while BAL27862-resistant tumour cells had IC₇₀ values >600 nanomolar.Paclitaxel and vinblastine data, using the same ex vivo assay, wasavailable for 7 of the 8 tumour models. Of these 7 models, all wereresistant to treatment with paclitaxel, while 6 were sensitive totreatment with vinblastine.

TABLE 1 Response to IC₇₀ BAL27862 Response to Response to Cancer typename BAL27862 [micromolar] paclitaxel vinblastine Gastric GFX 251sensitive 0.485 resistant sensitive Gastric GFX 97 resistant >3.5resistant sensitive Lung LXFE 211 sensitive 0.021 resistant sensitiveLung LXFE 397 resistant >3.5 Not known Not known Melanoma MEXF 1341sensitive 0.025 resistant sensitive Melanoma MEXF 276 resistant >3.5resistant sensitive Colorectal cancer CXF 1103 sensitive 0.022 resistantresistant Colorectal cancer CXF 243 resistant 0.696 resistant sensitiveImmunoblotting analysis was performed in order to measure the levels ofphospho-Akt (using an antibody recognising phosphorylated serine-473)and Akt protein in the same tumours maintained as xenografts. The actinlevels were included on the immunoblot as a loading control.Analysis of phospho-Akt levels indicated that phospho-Akt levels variedacross the tumours (FIG. 7).

Based on the colony outgrowth assay and the same IC₇₀ criteria, therewas no association between paclitaxel or vinblastine resistance and highphospho-Akt expression levels (compare FIG. 7 with Table 1). This isevident, for example, in the gastric cancer models. Although GXF 251 andGXF 97 were both resistant to paclitaxel, for GXF 251 the phospho-Aktlevels were virtually undetectable, while for GXF 97 the levels wereclearly higher. The same lack of association was true for the vincaalkaloid, vinblastine, in the gastric models, since both these tumourswere sensitive to vinblastine. This lack of association was repeated inthe melanoma and colorectal cancer models. Thus phospho-Akt levels wereshown to be unsuitable as a reliable biomarker of resistance to theconventional microtubule agents paclitaxel and vinblastine inpatient-derived tumour models.

Surprisingly, in contrast, when the BAL27862 resistance data, as definedby the colony outgrowth assay, was compared with the phospho-Akt level,phospho-Akt expression was shown to be higher only in the resistanttumours and not in the sensitive tumours derived from the same tumourhistotype (compare FIG. 7 with Table 1). Increased expression levelswere therefore consistently indicative of resistance to BAL27862. Thusphospho-Akt levels were shown to be a biomarker of resistance for thecompound according to the invention, BAL27862.

Example 4 Immunohistochemical Analysis of Gastric Tumour Xenografts

Immunohistocehmical analysis was performed on the gastric tumourxenografts (FIG. 8), revealing a higher level of phospho-Akt in thetumour model GXF 97. Again a clear correlation was seen between higherlevels of phospho-Akt and resistance to BAL27862 (tumour model GXF 97was BAL27862-resistant, while tumour model GXF 251 wasBAL27862-sensitive; as defined by the colony outgrowth assay—Table 1).Thus phospho-Akt levels were again shown to be a biomarker of resistancefor the compound according to the invention, BAL27862.

LIST OF ABBREVIATIONS

-   A549 human non-small cell lung cancer cell line-   BCA bicinchoninic acid-   BrdU bromodeoxyuridine-   BSA bovine serum albumin-   CA-125 cancer antigen 125-   CCD charge-coupled device-   CREST limited scleroderma syndrome-   DAB 3,3-diaminobenzidine-   DMSO dimethylsulphoxide-   DNA deoxyribonucleic acid-   dUTP 2′-Deoxyuridine 5′-Triphosphate-   EDTA ethylenediaminetetraacetic acid-   EGTA ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid-   ELISA enzyme-linked immunosorbent assay-   ESI-MS electrospray ionization mass spectrometry-   EtOH ethanol-   FACS fluorescence activated cell scan/sorting-   FCS/FBS foetal calf/foetal bovine serum-   G2/M transition from G2 to the mitotic phase in the cell cycle-   HeLa human squamous cell cancer cell line-   HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulphonic acid-   Hoe33342    2′-(4′-Ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5′-bis-1H-benzimidazole    trihydrochloride trihydrate-   IC₇₀ concentration at which 70% signal is inhibited-   IgG immunoglobulin G-   IHC immunohistochemistry-   ISET isolation by size of epithelial tumor cells-   MALDI matrix-assisted-laser-desorption/ionisation mass-spectrometry-   mTORC2 mammalian target of rapamycin complex2-   MTS    3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium-   NaF sodium fluoride-   NCBI National Center for Biotechnology Information-   NSCLC non-small cell lung cancer-   NP40 Nonidet P40-   PBS phosphate buffered saline-   P-gp P-glycoprotein-   PKB protein kinase B-   PMSF phenylmethylsulphonyl fluoride-   PSA prostate-specific antigen-   PVDF polyvinylidene fluoride-   RAC related to A and C kinases-   RANO response assessment for high-grade gliomas-   RECIST response evaluation criteria in solid tumors-   RICTOR Rapamycin-insensitive companion of mTOR-   RPMI-1640 cell culture medium used for culturing transformed and    non-transformed eukaryotic cells and cell lines-   SDS sodium dodecyl sulphate-   SEQ. ID No. sequence identification number-   siRNA small inhibitory ribonucleic acid-   SKBR3 human mammary carcinoma cell line-   TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling-   Tween non-ionic detergent-   YO-PRO fluorescent, monomeric cyanine, nucleic acid stain

1-25. (canceled)
 26. A method of determining whether a subject isresistant to treating a disease by administration to said subject acompound of formula I

wherein R represents phenyl, thienyl or pyridinyl wherein phenyl isoptionally substituted by one or two substituents independently selectedfrom alkyl, halo-lower alkyl, hydroxy-lower alkyl, lower alkoxy-loweralkyl, acyloxy-lower alkyl, phenyl, hydroxy, lower alkoxy, hydroxy-loweralkoxy, lower alkoxy-lower alkoxy, phenyl-lower alkoxy, loweralkylcarbonyloxy, amino, monoalkylamino, dialkylamino, loweralkoxycarbonylamino, lower alkylcarbonylamino, substituted amino whereinthe two substituents on nitrogen form together with the nitrogenheterocyclyl, lower alkylcarbonyl, carboxy, lower alkoxycarbonyl, cyano,halogen, and nitro; and wherein two adjacent substituents aremethylenedioxy; and wherein pyridinyl is optionally substituted by loweralkoxy, amino or halogen; wherein Y is oxygen or nitrogen substituted byhydroxy or lower alkoxy; or when R¹ is phenyl or pyridinyl, X isadditionally oxygen, R¹ represents hydrogen, lower alkylcarbonyl,hydroxy-lower alkyl or cyano-lower alkyl; R², R³ and R⁶ representhydrogen; R⁴ and R⁵, independently of each other, represent hydrogen,lower alkyl or lower alkoxy; or R⁴ and R⁵ together representmethylenedioxy; wherein said prefix denotes a radical having up to 7carbon atoms; or pharmaceutically acceptable derivatives thereof; saidmethod comprising; a) obtaining a sample from said subject; b) measuringthe level of the phospho-Akt proteins in said sample, said phospho-Aktproteins being selected from the group consisting of phospho-Akt1,phospho-Akt2; and phospho-Akt3, wherein the phospho-Akt proteins containat least one phosphorylation at a site other than T308 of phospho-Akt1,T309 of phospho-Akt2 and T305 of phospho-Akt3 respectively; and c)thereby determining from said measured level of the phospho-Akt proteinswhether the subject is resistant to treating of said disease with saidcompound of formula I.
 27. The method of claim 26 wherein thephospho-Akt proteins are also phosphorylated at site T308 ofphospho-Akt1, T309 of phospho-Akt2 and T305 of and phospho-Akt3respectively.
 28. The method of claim 26, wherein said subject is ahuman or animal, said sample is taken from the human or animal body, andthe level of phospho-Akt proteins in said sample is measured ex vivo.29. The method of claim 28, wherein the sample is derived from tumourtissue, normal tissue, circulating tumour cells, cell lines, plasma,whole blood or serum.
 30. The method of claim 29, wherein the subjectsare human.
 31. The method of claim 30, wherein the protein sequence ofsaid phospho-Akt proteins is selected from the group consisting of SEQID No. 1, SEQ ID 2, SEQ ID 3 and homologues, mutant forms, allelicvariants, isoforms, splice variants and proteins with sequences havingat least 75% identity to SEQ ID 1, SEQ ID 2, or SEQ ID
 3. 32. The methodof claim 31, wherein said phospho-Akt proteins are also phosphorylatedat site T308 of SEQ ID1, T309 of SEQ ID2 and T305 of SEQ ID3respectively.
 33. The method of claim 31, wherein said phospho-Aktproteins are phosphorylated on the following serine residue: for SEQID1: S473; for SEQ ID2: S474; and for SEQ-ID3: S472.
 34. The method ofclaim 33, wherein said phospho-Akt proteins are also phosphorylated onthe following threonine residue: for SEQ ID1: T308; for SEQ ID2: T309;and for SEQ ID3: T305.
 35. The method of claim 28, wherein thedetermination of a higher level of phospho-Akt proteins in the samplefrom a subject relative to a standard value or values of phospho-Aktprotein levels predicts resistance to treating said disease with saidcompound of formula I.
 36. The method of claim 33, wherein the proteinsequence of the phospho-Akt is selected from the group consisting of SEQID No. 1, SEQ ID 2, SEQ ID 3, and homologues, mutant forms, allelicvariants, isoforms, splice variants and proteins with sequences havingat least 75% identity to SEQ ID 1, SEQ ID 2 or SEQ ID
 3. 37. The methodof claim 35, wherein the measured phospho-Akt protein levels in saidsample is determined by comparing the measured phospho-Akt proteinlevels in said sample with: i) a standard value or a set of standardvalues of phospho-Akt protein levels from samples of subjects; or ii) astandard value or a set of standard values of phospho-Akt protein levelsfrom normal cells, tissues or body fluids.
 38. The method of claim 28wherein the diseases is disease is selected from the group consisting ofbreast cancer, prostate cancer, cervical cancer, ovarian cancer, gastriccancer, colorectal cancer, pancreatic cancer, liver cancer, braincancer, neuroendocrine cancer, lung cancer, kidney cancer, hematologicalmalignancies, melanoma and sarcomas and the sample is derived from tumortissue or circulating tumor cells.
 39. The method of claim 35, whereinthe measured phospho-Akt protein levels in said sample is determined bycomparing the measured phospho-Akt protein levels in said sample with:i) a standard value or a set of standard values of phospho-Akt proteinlevels from samples of subjects with the same tumor histotype; or ii) astandard value or a set of standard values of phospho-Akt protein levelsfrom normal tissues or cells.
 40. The method of claim 26, wherein thecompound is a compound of general formula I wherein R represents phenylor pyridinyl wherein phenyl is optionally substituted by one or twosubstituents independently selected from lower alkyl, lower alkoxy,amino, acetylamino, halogen and nitro; and wherein pyridinyl isoptionally substituted by amino or halogen; X represents a group C═O; R¹represents hydrogen or cyano-lower alkyl; R², R³, R⁴, R⁵ and R⁶represent hydrogen; or pharmaceutically acceptable derivatives thereof.41. The method of claim 26, wherein the compound is represented by thefollowing formula

wherein R, Y and R¹ are defined as follows: R Y R¹

O CH₂CH₂CN

O H

O CH₂CH₂CN

or pharmaceutically acceptable derivatives thereof.
 41. The method ofclaim 26, wherein the compound is

or pharmaceutically acceptable derivatives thereof.
 43. The method ofclaim 26 wherein a pharmaceutically acceptable derivative is selectedfrom the group consisting of a salt, solvate, pro-drug, salt of apro-drug, polymorph and isomer of the compound of general formula I. 44.The method of claim 43, wherein the pro-drug is an amide formed from anamino group present within the R group and the carboxy group is glycine,alanine or lysine.
 45. The method of claim 26, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 46. A method forpredicting the response to treatment of a disease in a patient subjectby administration of a compound of general formula I

wherein R represents phenyl, thienyl or pyridinyl wherein phenyl isoptionally substituted by one or two substituents independently selectedfrom the group consisting of alkyl, halo-lower alkyl, hydroxy-loweralkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, phenyl, hydroxy,lower alkoxy, hydroxy-lower alkoxy, lower alkoxy-lower alkoxy,phenyl-lower alkoxy, lower alkylcarbonyloxy, amino, monoalkylamino,dialkylamino, lower alkoxycarbonylamino, lower alkylcarbonylamino,substituted amino wherein the two substituents on nitrogen form togetherwith the nitrogen heterocyclyl, lower alkylcarbonyl, carboxy, loweralkoxycarbonyl, cyano, halogen, and nitro; and wherein two adjacentsubstituents are methylenedioxy; and wherein pyridinyl is optionallysubstituted by lower alkoxy, amino or halogen; wherein Y is oxygen ornitrogen substituted by hydroxy or lower alkoxy; or when R1 is phenyl orpyridinyl, X is additionally oxygen, R¹ represents hydrogen, loweralkylcarbonyl, hydroxy-lower alkyl or cyano-lower alkyl; R², R³ and R⁶represent hydrogen; R⁴ and R⁵, independently of each other, representhydrogen, lower alkyl or lower alkoxy; or R⁴ and R⁵ together representmethylenedioxy; or pharmaceutically acceptable derivatives thereof, andwherein said prefix lower denotes a radical having up to 7 carbon atoms,said method comprising the steps of: a) measuring the level of thephospho-Akt proteins in said sample, said phospho-Akt proteins beingselected from the group consisting of phospho-Akt1, phospho-Akt2; andphospho-Akt3, wherein the phospho-Akt proteins contain at least onephosphorylation at a site other than T308 of phospho-Akt1, T309 ofphospho-Akt 2, and T305 of phospho-Akt3 respectively; and b) comparingthe value or values of the levels from step a) with a standard value ora set of standard values which comparison is predictive ofresponsiveness to compounds of formula I.
 47. The method of claim 26wherein the phospho-Akt proteins are also phosphorylated on thefollowing threonine residue: for phospho-Akt1: T308; for phospho-Akt2:T309; and for phospho-Akt3: T305.
 48. The method of claim 46, whereinsaid patient is an animal or human being and the level of phospho-Aktproteins is measured ex vivo in the sample taken from said animal orhuman being.
 49. The method according to claim 46, wherein the sample isderived from normal tissue, tumor tissue, circulating tumor cells,plasma or whole blood.
 50. The method of claim 49, wherein a higherlevel of phospho-Akt proteins in the sample relative to a standard valueor a set of standard values predicts resistance to treating said diseasewith said compound of formula I.
 51. The method of claim 50, wherein thedetermination of a higher level of phospho-Akt in said sample obtainedfrom the animal or human being is carried out by comparing the measuredphospho-Akt protein level in said sample i) relative to a standard valueor a set of standard values of levels of phospho-Akt proteins fromsamples; or ii) relative to a standard value or a set of standard valuesof levels of phospho-Akt proteins from a sample or samples of levels ofphospho-Akt from normal cells, tissues or body fluids.
 52. The method ofclaim 51, wherein the samples are human.
 53. The method of claim 52,wherein the protein sequence of phospho-Akt is selected from the groupconsisting of SEQ ID No. 1, SEQ ID 2, SEQ ID 3 and homologues, mutantforms, allelic variants, isoforms, splice variants and proteins withsequences having at least 75% identity to SEQ ID 1, SEQ ID 2, or SEQ ID3.
 54. The method of claim 53, wherein the phospho-Akt has beenphosphorylated on the following serine residue: for SEQ ID 1: S473; forSEQ ID 2: S474; and for SEQ ID 3: S472.
 55. The method of claim 46,wherein the compound is a compound of general formula I wherein Rrepresents phenyl or pyridinyl wherein phenyl is optionally substitutedby one or two substituents independently selected from the groupconsisting of lower alkyl, lower alkoxy, amino, acetylamino, halogen andnitro; and wherein pyridinyl is optionally substituted by amino orhalogen; X represents a group C═O; represents hydrogen or cyano-loweralkyl; R², R³, R⁴, R⁵ and R⁶ represent hydrogen; or pharmaceuticallyacceptable derivatives thereof, and wherein said prefix lower denotes aradical having up to and including a maximum of
 7. 56. The method ofclaim 50, wherein said disease is a cancer.
 57. The method according toclaim 56, wherein the sample is derived from normal tissue, tumortissue, circulating tumor cells, plasma or whole blood.
 58. The methodof claim 57, wherein the determination of a higher level of phospho-Aktin said sample obtained from the animal or human being is carried out bycomparing the measured phospho-Akt protein level in said sample i)relative to a standard value or a set of standard values of levels ofphospho-Akt proteins from samples from other subjects having the sametumor histotype as said animal or human being; or ii) relative to astandard value or a set of standard values of levels of phospho-Aktproteins from a sample or samples of levels of phospho-Akt proteins fromnormal tissue.
 59. The method of claim 58, wherein the phospho-Akt isused as biomarker to select subjects suffering or predisposed tosuffering from a disease for treatment with a compound of generalformula I or pharmaceutically acceptable derivatives thereof.
 60. Themethod of claim 59, wherein the phospho-Akt proteins are used asbiomarker to select subjects suffering or predisposed to suffering fromcancer for treatment with a compound of general formula I orpharmaceutically acceptable derivatives thereof.
 61. The method of claim46, wherein the compound is represented by the following formula

wherein R, Y and R1 are defined as follows: R Y R¹

O CH₂CH₂CN

O H

O CH₂CH₂CN

or pharmaceutically acceptable derivatives thereof.
 62. The method ofclaim 46, wherein the compound is

or pharmaceutically acceptable derivatives thereof.
 63. The method ofclaim 62, wherein a pharmaceutically acceptable derivative is selectedfrom the group consisting of a salt, solvate, pro-drug, salt of apro-drug, polymorph and isomer of the compound of general formula I. 64.The method of claim 63, wherein the pharmaceutically acceptable pro-drugis an amide formed from an amino group present within the R group of thecompound of formula I as defined in claim 46 and the carboxy group ofglycine, alanine or lysine.
 65. The method of claim 55 wherein apharmaceutically acceptable derivative is selected from the groupconsisting of a salt, solvate, pro-drug, salt of a pro-drug, polymorphand isomer of the compound of general formula I as defined in claim 52.66. The method of claim 65, wherein the pharmaceutically acceptablepro-drug is an amide formed from an amino group present within the Rgroup of the compound of formula I as defined in claim 46 and thecarboxy group of glycine, alanine or lysine.
 67. The method of claim 46,wherein the compound is

or a pharmaceutically acceptable salt thereof.
 68. The method of claim46, wherein the disease is a neoplastic disease or autoimmune disease.69. The method of claim 68 wherein the disease is selected from thegroup consisting of breast cancer, prostate cancer, cervical cancer,ovarian cancer, gastric cancer, colorectal cancer, pancreatic cancer,liver cancer, brain cancer, neuroendocrine cancer, lung cancer, kidneycancer, hematological malignancies, melanoma and sarcomas.
 70. Themethod of claim 69, wherein the disease is selected from the groupconsisting of breast cancer, cervical cancer, gastric cancer, lungcancer and melanoma.
 71. The method of claim 70, wherein the disease isselected from the group consisting of gastric cancer, lung cancer andmelanoma.
 72. The method of claim 70, wherein said disease is anautoimmune disease.
 73. The method of claim 72, wherein said autoimmunedisease is systemic, discoid or subacute cutaneous lupus erythematosus,rheumatoid arthritis, antiphospholipid syndrome, CREST, progressivesystemic sclerosis, mixed connective tissue disease (Sharp syndrome),Reiter's syndrome, juvenile arthritis, cold agglutinin disease,essential mixed cryoglobulinemia, rheumatic fever, ankylosingspondylitis, chronic polyarthritis, myasthenia gravis, multiplesclerosis, chronic inflammatory demyelinating polyneuropathy,Guillan-Barre syndrome, dermatomyositis/polymyositis, autoimmunehemolytic anemia, thrompocytopenic purpura, neutropenia, type I diabetesmellitus, thyroiditis (including Hashimoto's and Grave's disease),Addison's disease, polyglandular syndrome, pemphigus (vulgaris,foliaceus, sebaceous and vegetans), bullous and cicatricial pemphigoid,pemphigoid gestationis, epidermolysis bullosa acquisita, linear IgAdisease, lichen sclerosus et atrophicus, morbus Duhring, psoriasisvulgaris, guttate, generalized pustular and localized pustularpsoriasis, vitiligo, alopecia greata, primary biliary cirrhosis,autoimmune hepatitis, all forms of glomerulonephritis, pulmonalhemorrhage (goodpasture syndrome), IgA nephropathy, pernicious anemiaand autoimmune gastritis, inflammatory bowel diseases (including colitisulcerosa and morbus Crohn), Behcet's disease, Celic-Sprue disease,autoimmune uveitis, autoimmune myocarditis, granulomatous orchitis,aspermatogenesis without orchitis, idiopatic and secondary pulmonaryfibrosis, inflammatory diseases with a possibility of autoimmunepathogensesis, such as pyoderma gangrensosum, lichen ruber, sarcoidosis(including Lofgren and cutaneous/subcutaneous type), granuloma anulare,allergic type I and type IV immunolgical reaction, asthma bronchiale,pollinosis, atopic, contact and airborne dermatitis, large vesselvasculitis (giant cell and Takayasu's arteritis), medium sized vesselvasculitis (polyarteritis nodosa, Kawasaki disease), small vesselvasculitis (Wegener's granulomatosis, Churg Strauss syndrome,microscopic polangiitis, HenochSchoenlein purpura, essentialcryoglobulinemic vasculitis, cutaneous leukoklastic angiitis),hypersensitivity syndromes, toxic epidermal necrolysis (Stevens-Johnsonsyndrome, erythema multiforme), diseases due to drug side effects, allforms of cutaneous, organ-specific and systemic effects due to type 1-vu(Coombs classification) immunologic forms of reaction, transplantationrelated pathologies, such as acute and chronic graft versus host andhost versus graft disease, involving all organs (skin, heart, kidney,bone marrow, eye, liver, spleen, lung, muscle, central and peripheralnerve system, connective tissue, bone, blood and lymphatic vessel,genito-urinary system, ear, cartillage, primary and secondary lymphaticsystem including bone marrow, lymph node, thymus, gastrointestinaltract, including oro-pharynx, esophageus, stomach, small intestine,colon, and rectum, including parts of above mentioned organs down tosingle cell level and substructures, e.g. stem cells).
 74. The method oftreating a neoplastic or autoimmune disease in a patient in needthereof, said method comprising a) obtaining a sample of biologicmaterial from the body of said patient; b) determining the level of thephospho-Akt proteins in said sample said phospho-Akt proteins beingselected from the group consisting of phospho-Akt1, phospho-Akt2; andphospho-Akt3, wherein the phospho-Akt proteins contain at least onephosphorylation at a site other than T308 of phospho-Akt1, T309 ofphospho-Akt2, and T305 of phospho-Akt3 respectively; and c) thentreating the patient with a compound of formula I as described above ora pharmaceutically acceptable derivative thereof, if the level ofphospho-Akt proteins in said sample is not higher than a standard valueor set of standard values for the level of phospho-Akt proteins.
 75. Themethod of claim 74, wherein said neoplastic disease is cancer.
 76. Themethod of claim 75 wherein the phospho-Akt proteins are alsophosphorylated at site T308 of phospho-Akt1, T309 of phospho-Akt2 andT305 of and phospho-Akt3 respectively.
 77. The method of claim 76,wherein the standard values of phospho-Akt protein are determined i)from samples of other subjects having the same tumour histotype as saidanimal or human being; or ii) from a sample or samples of normal tissue.78. A kit for predicting the response to a compound of general formula Ior a pharmaceutically acceptable derivative thereof, as defined in claim41 comprising reagents necessary for measuring the level of phospho-Aktproteins in a sample, said phospho-Akt proteins being selected from thegroup consisting of phospho-Akt1, phospho-Akt2; and phospho-Akt3,wherein the phospho-Akt proteins contain at least one phosphorylation ata site other than T308 of phospho-Akt1, T309 of phospho-Akt2 and T305 ofand phospho-Akt3 respectively and a comparator module which comprises astandard value or set of standard values to which the level ofphospho-Akt proteins in the sample is compared.
 79. The kit according toclaim 78, wherein the reagents comprise: a) a capture reagent comprisinga detector for phospho-Akt proteins and b) a detection reagent.
 80. Thekit according to claim 79, wherein said capture reagent is an antibody.81. The kit according to claim 80, wherein the kit comprises a compoundof the following formula or a pharmaceutically acceptable salt thereof,


82. The kit of claim 81, wherein said salt is a hydrochloride salt. 83.A device for predicting the response to a compound of general formula Ior a pharmaceutically acceptable derivative thereof, as defined in claim43, comprising: reagents necessary for measuring the level of thephospho-Akt proteins in a sample, said phospho-Akt proteins beingselected from the group consisting of phospho-Akt1, phospho-Akt2; andphospho-Akt3, wherein the phospho-Akt proteins contain at least onephosphorylation at a site other than T308 of phospho-Akt1, T309 ofphospho-Akt2 and T305 of and phospho-Akt3 respectively and a comparatormodule which comprises a standard value or set of standard values towhich the level of phospho-Akt in the sample is compared.
 84. A methodfor treating a neoplastic or autoimmune disease in a subject patient,that has a sample with a higher level of phospho-Akt proteins comparedto a standard level or set of standard levels, said phospho-Akt proteinsbeing selected from the group consisting of phospho-Akt1, phospho-Akt2;and phospho-Akt3, wherein the phospho-Akt proteins contain at least onephosphorylation at a site other than T308 of phospho-Akt1, T309 ofphospho-Akt2 and T305 of and phospho-Akt3 respectively, by firstdecreasing the level of phospho-Akt proteins in said patient to saidstandard level or set of standard levels or a level below and thereaftertreating said subject patient with a compound of formula I or apharmaceutically acceptable derivative thereof as referred to in claim43.
 85. The method of claim 84, wherein said disease is cancer.